bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polysome Translational State during the Cell Cycle
Authors: EREMENKO Tamilla , VOLPE Pietro 
Published: March 2005

HeLa cells were synchronized with a double thymidine block. Ribosomal subunits, monomers and polyribosomes have been quantitatively analysed at hourly intervals, during interphase, and every 15 min, during mitosis. This analysis was performed on linear 7–47% sucrose gradients.From the beginning of G1 up to the end of S phase, a certain equilibrium among ribosomal sub‐units, monomers and polyribosomes is maintained, while from the time of entering G2 to M the translation machinery appears to be mobilized in the sense of polysome formation. Under these conditions, the amount of polysomes per cell during the mitotic cycle is expressed by a bi‐phasic pattern showing pre‐ and post‐mitotic peaks with a falling‐off during S. The G1 peak, meanwhile, is much lower than the G2 peak.The incorporation of [3H]leucine into nascent polypeptide chains on polysomes, as well as into bulk cell proteins and into nuclear and cytoplasmic proteins considered separately, is also represented by a bi‐phasic curve which shows, however, a higher peak in G1 and a lower peak in G2, with two fallings‐off during S and M, respectively. Since between the G1 and the G2 amino acid pools there are not strong differences of leucine concentration, the discrepancy between the amount of polysomes and the rate of labelling is discussed on the basis of the differences of polysome shape found at the different stages of the cycle. In young cells, in fact, there is an abundance of small polysomes, while in the old cell large polysomes predominate.It is suggested that, in the old cell, the rate of translation on large polysomes could be relatively lower or that among these heavy aggregates a given number of “frozen” polysomes could be present. The ribosome state is considered as a probable limiting‐factor of translation, particularly in mitosis.
Studies of Glutamate Dehydrogenase: Analysis of Functional Areas and Functional Groups
Authors: HUCHO Ferdinand , RASCHED Ihab , SUND Horst 
Published: March 2005

It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End‐group analysis after trypsin treatment yields aspartic acid as the new N‐terminal amino acid while the C‐terminal threonine remains unchanged.
NADH, especially in the presence of 2‐oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated.
The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed.
The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N‐terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished.
It is shown by treatment of the enzyme with iodo[2‐14C]acetic acid as well as with Ellman's reagent that the six –SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rate of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme.
Cross‐linking of the enzyme molecule with bifunctional reagents and subsequent dodecyl‐sulfate‐polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three.
The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.
Bacterial Respiration‐Linked Proton Translocation and Its Relationship to Respiratory‐Chain Composition
Authors: JONES Colin W. , BRICE Joan M. , DOWNS Andrew J. , DROZD Jan W. 
Published: March 2005

The relationship between respiratory chain composition and the efficiency of respiration‐linked proton translocation was studied in nine bacterial species of widely differing taxonomic and ecological status.
All the bacteria investigated contained respiratory chain dehydrogenases, ubiquinone and/or menaquinone, cytochrome b and cytochrome oxidase aa3 and/or o. In addition, some of these organisms also contained pyridine nucleotide transhydrogenase and/or cytochrome c.
→H+/O ratios of whole cell suspensions oxidising endogenous substrates were in the approximate range 4–8 mol H+ translocated per g‐atom oxygen consumed. It was concluded from the observed →+H+/O ratios of cells loaded with specific substrates that proton‐translocating loops 1 and 2 were present in all of the organisms investigated, but that loops 0 and 3 were dependent upon the presence of pyridine nucleotide transhydrogenase and cytochrome c respectively.
The wide range in energy conservation efficiency which was observed in these organisms is discussed in relation to their respiratory chain composition and natural habitat.
Isoenzymes of p‐Coumarate: CoA Ligase from Cell Suspension Cultures of Glycine max
Authors: KNOBLOCH Karl‐Heinz , HAHLBROCK Klaus 
Published: March 2005

Two isoenzymes of p‐coumarate: CoA ligase were isolated from cell suspension cultures of soybean (Glycine max L., var. Mandarin). Separation and partial purification of the enzymes were achieved by precipitation with MnCl2 and (NH4)2SO4 and column chromatography on DEAE‐cellulose, Sephadex G‐100 and hydroxyapatite.The isoenzymes had approximately the same molecular weight, but differed significantly with respect to their substrate specificity, their inhibition constants for AMP, their dependence on pH and ionic strength for optimum activity, and their fractionation pattern during the purification procedure or upon analytical disc‐gel electrophoresis.Both coumarate: CoA ligases were specific for the activation of various substituted cinnamic acids. Of the cinnamic acids tested, ferulic, sinapic, 5‐hydroxyferulic, p‐coumaric, and caffeic acids were the substrates with the lowest apparent Km values (all on the order of 1 to 4 × 10−5 M) for isoenzyme 1. The lowest apparent Km values (from about 1 to 9 × M) for isoenzyme 2 were obtained for caffeic, p‐coumaric, m‐coumaric, and o‐coumaric acids. Sinapic acid and several methoxycinnamic acids were efficient substrates of isoenzyme 1 but were not activated at all by isoenzyme 2. The possible roles of the two p‐coumarate: CoA ligase isoenzymes in the phenylpropanoid metabolism of the cell cultures are discussed.
Studies of Lipopolysaccharides from Pseudomonas aeruginosa
Authors: WILKINSON Stephen G. , GALBRAITH Lesley 
Published: March 2005

Lipopolysaccharides from 13 strains of Pseudomonas aeruginosa representing seven serotypes of the Habs scheme have been analysed. The lipid A fractions, obtained by mild acid hydrolysis of the lipopolysaccharides, contained phosphorylated glucosamine residues substituted with dodecanoic, hexadecanoic, 2‐hydroxydodecanoic, 3‐hydroxydecanoic, and 3‐hydroxydodecanoic acids (hexadecanoic acid and 2‐hydroxydodecanoic acid were absent from one lipid A). Low‐molecular‐weight solutes released during the mild hydrolyses included 2‐keto‐3‐deoxyoctonic acid, inorganic ortho‐phosphates and pyrophosphates, ethanolamine mono, pyro and triphosphates. For most strains two polysaccharide fractions, one of which appeared to be the common core polysaccharide, were obtained. The major identifiable components and their approximate proportions in the core polysaccharides were glucose (3–4), rhamnose (1), galactosamine (1), alanine (1–1.5), phosphorus (4–6) And heptose (1–2). Rhamnose was absent from one polysaccharide; another lacked both rhamnose and alanine but contained glucosamine. Small amounts of various amino sugars found in other core polysaccharides could be associated with the presence of higher‐molecular‐weight material. Such material was isolated from strain NCIB 8626.The high‐molecular‐weight polysaccharides obtained from ten strains were probably heterogeneous and consisted mainly of amino compounds, though rhamnose was a major component of four polysaccharides and arabinose was present in another. Fucosamine was the most common amino sugar, but quinovosamine, glucosamine, galactosamine, a possible aminohexuronic acid and unidentified amino compounds were also detected.The results of the survey are discussed in terms of the serological classification of the bacteria and of their sensitivity to EDTA.
Muscle Pyruvate Kinase: Interaction with Substrates and Analogues Studied by Difference Spectroscopy
Authors: BREVET Annie , ROUSTAN Claude , PRADEL Louise‐Anne , VAN THOAI Nguyen 
Published: March 2005

The substrate binding sites of pyruvate kinase have been studied by means of spectrophotometric investigations. Two binding sites, one for the nucleotide substrate and one for the acceptor, have been characterized.The interaction of nucleotide substrates with the enzyme, which is metal‐dependent, results in a perturbation of the spectrum of the nucleotide chromophore characterized by hypochromic and red shift effects; the hydrophobicity of the nucleotide site was estimated by using a reporter group reagent, 2‐(dansylamino)ethyl monophosphate.The interaction of pyruvate or phosphoenolpyruvate with pyruvate kinase induces perturbation of a tyrosyl chromophore.The comparison between the binding sites of several ATP phosphotransferases is discussed and some common features are reported.
N‐Acetylglutamate 5‐Phosphotransferase of Pseudomonas aeruginosa
Authors: HAAS Dieter , LEISINGER Thomas 
Published: March 2005

N‐Acetylglutamate 5‐phosphotransferase (ATP: N‐acetyl‐l‐glutamate 5‐phosphotransferase EC 2.7.2.8) the second enzyme of arginine biosynthesis, was purified over 2000‐fold from Pseudomonas aeruginosa. The purification procedure involved a heat treatment, ammonium sulfate precipitation, and chromatography on DEAE‐cellulose, Sephadex G‐150, and hydroxyapatite. The purified enzyme was greater than 90% pure as judged by analytical polyacrylamide gel electrophoresis. A molecular weight of approximately 230000 was obtained by gel filtration. Electrophoresis in sodium dodecyl sulfate gels gave a single band corresponding to a molecular weight of 29000.Due to the capacity for self‐association, the enzyme can exist in different states of aggregation depending on the nature of ligands and the concentrations of phosphate buffer. As estimated by gel filtration, the molecular weight was about 230000 in the presence of N‐acetyl‐l‐glutamate. With l‐arginine, the feedback inhibitor, and MgATP forms of smaller molecular weight (minimum of approximately 65000) were found. A concurrent change in the sedimentation coefficient as a function of ligands was demonstrated by sucrose gradient centrifugation.The synthesis of N‐acetylglutamate 5‐phosphotransferase was not repressed by exogenous l‐arginine or its precursors.
Unspecific Arginine Kinase of Molecular Weight 150000. Amino Acid Composition, Subunit Structure and Number of Substrate Binding Sites
Authors: ROBIN Yvonne , GUILLOU Annie , VAN THOAI Nguyen 
Published: March 2005

The amino acid composition of unspecific arginine kinase of molecular weight 150000 of Sabella pavonina muscle has been determined. It was found to be very similar to that of the phosphagen kinases previously studied.The subunit structure of the enzyme has been investigated by physical and chemical means. The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle arginine kinase is composed of four non‐covalently linked polypeptide chains, with similar molecular weights.The number of binding sites for the nucleotide substrate ADP‐Mg2+ has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150000.Thus, arginine kinase from Sabella muscle, of molecular weight 150000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.
Digestion with Matrix‐Bound Proteases as a Possible Probe for the Topography of the DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: LILL Helmut R. , HARTMANN Guido R. 
Published: March 2005

DNA‐dependent RNA polymerase lacking subunit à was digested with matrix‐bound chymotrypsin or trypsin in the presence of 0.4 M NaCI in the monomeric form or at low ionic strength in the oligomeric form. Sigma‐containing polymerase was digested in the same way. The course of proteolysis was followed by polyacrylamide gel electrophoresis after dissociation of the enzyme with detergent into subunits and the fragments produced by the hydrolysis. The following results were obtained.(a)The large subunits β and β′ are cleaved with a much higher rate in the monomeric than in the oligomeric polymerase. (b) Both large subunits are hydrolysed with the same rate. (c) Subunit α is hydrolysed almost with the same rate in the monomeric and oligomeric form of polymerase. (d) The same was found for subunit à. (e) These effects were independent of the substrate specificity of the protease used. (f) Subunit à is much more susceptible to chymotrypsin than to trypsin. (g) Subunit à protects the large subunits β and β′ against tryptic cleavage.These results can be explained in terms of a tentative model for the topography of the protomer‐protomer interactions in RNA polymerase. According to this model subunits β and β′ contain two sites for isologous interactions of protomers. One site can be blocked by attachment of subunit à. Subunits α and à do not participate directly in the association.
Characterization of a Specific Transport System for Arginine in Isolated Yeast Vacuoles
Authors: BOLLER Thomas , DÜRR Mathias , WIEMKEN Andres 
Published: March 2005

The transport of l‐arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured.Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts.The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined.From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol.Arginine transport shows saturation kinetics with an apparent Km of 30 μM in the isolated vacuoles, and of 1.5 μM in the spheroplasts.Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities.The inhibition of vacuolar l‐arginine transport by d‐arginine, l‐histidine, and l‐canavanine was competitive with apparent Ki values of 60 μM, 400 μM and 600 μM respectively.
No Correlation between Native and Denatured Forms of tRNATrp from Escherichia coli and the Resistant and Sensitive Molecules Characterised by Phosphorolysis
Authors: THANG Minh Nguy , BUCKINGHAM Richard Hugh , DONDON Lilian 
Published: March 2005

Some tRNA molecules in solution are sensitive to attack by polynucleotide phosphorylase while others are resistant, even with pure species of tRNA. Further analysis of this behaviour has revealed an underlying microheterogeneity in tRNA structure.In order to clarify the relation between the sensitive and resistant classes of tRNA, and the native and denatured forms with respect to amino acid acceptance, the phosphorolysis of tRNATrp from Escherichia coli has been investigated. Native tRNATrp is similar to species examined previously: resistant and sensitive classes are observed and the sensitive proportion increases with temperature. At 20°C both native and denatured tRNATrp are stable under phosphorolysis conditions, and denatured tRNATrp is found also to possess resistant and sensitive classes. About 10% of both native and denatured tRNATrp is rapidly phosphorolysed at 20°C, but the rate of conversion of resistant denatured tRNATrp to the sensitive class is about twice as fact as for the native form. Thus it can be concluded that the sensitive molecules of tRNATrp attacked by polynucleotide phosphorylase are not due to denaturation.
Identification of Two Products of Mitochondrial Protein Synthesis Associated with Mitochondrial Adenosine Triphosphatase from Neurospora crassa
Authors: JACKL Gerhard , SEBALD Walter 
Published: March 2005

Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecyl‐sulfate‐gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000.At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X‐100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F1 ATPase.Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis.The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex.
The Mitochondrial ATPase
Authors: FERGUSON Stuart J. , LLOYD William J. , RADDA George K. 
Published: March 2005

When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4‐chloro‐7‐nitrobenzofurazan, is incubated at pH 9.0, the 7‐nitrobenzofurazan group undergoes an intramolecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N‐nitrobenzofurazan ATPase has little or no activity.
The fluorescence of the N‐nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP.
Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N‐nitrobenzofurazan chromophore is exclusively in the β subunit.
The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds.
The interaction of the N‐nitrobenzofurazan ATPase with aurovertin is reported.
Photoinactivation and Carbethoxylation of Leucine Aminopeptidase
Authors: LUDEWIG Manfred , FROHNE Marlies , MARQUARDT Irmgard , HANSON Horst 
Published: March 2005

In the present paper the reactivity of histidyl residues of leucine aminopeptidase from bovine eye lens was studied by dye‐sensitized photooxidation and by carbethoxylation of the enzyme protein using diethylpyrocarbonate.Of all the different amino acids modified by photooxidation only histidine is connected with the enzymic activity, whereas tyrosine seems to be involved in structure stabilization.By changing the pH and varying the effectors (Mg2+ and/or dodecylsulfate) of the reaction mixture a different number of histidyl residues of the enzyme protein is caused to react with diethylpyrocarbonate. No secondary reactions with tyrosyl or tryptophyl residues could be observed by spectrophotometric investigations.The enzyme modified by one of the above‐mentioned methods shows changes in the capacity of Mn2+ binding measured by autoradiography as well as in the degree of enhancement of enzymic activity by Mn2+ or Mg2+ ions.Of the 48 histidyl residues of the enzyme (Mr= 326000) up to 2 histidyl residues per subunit (Mr= 54000) may be involved in Mn2+ or Mg2+ binding and up to 4 histidyl residues have a strong influence on Zn2+ binding.
The Effects of Spermine and Mg2+ on the Catalytic Mechanism of Isoleucine: tRNA Ligase
Authors: CARR Alison C. , PENZER Geoffrey R. , PLUMBRIDGE Jacqueline A. , IGLOI Gabor L. 
Published: March 2005

Isoleucyl‐tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH‐range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects.
The spermine‐stimulated reaction has a steeper temperature‐dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl‐tRNA formation are detected in the presence of Mg2+± spermine, but only a single step is observed in the presence of spermine alone. Thus the rate‐limiting steps under normal assay conditions are different for the two cations.
Enzyme‐bound isoleucyl‐AMP can be formed in the absence of Mg2+ and ± spermine.
It is concluded that there is no evidence for cation‐dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.
Surface Carbohydrate Composition of Different Types of Chicken Lymphocytes
Authors: DROEGE Wulf , STROMINGER Jack L. , SINGH Prem Pal , LÜDERITZ Otto 
Published: March 2005

A simple and quick procedure was used to analyse the carbohydrate composition of surface glycoproteins from chicken lymphocytes. The procedure included papain digestion of the cells, a two‐step purification of the supernatant material and a sugar analysis by gaschromatography. The method made it possible to analyse samples of about 109 lymphocytes.The surface glycoproteins from different chicken lymphocyte preparations were found to differ significantly in their sugar composition. Lymphocytes from thymus, bursa, spleen or blood were characterized by typical relative amounts of glucose, galactosamine, fucose and sialic acid. The values for mannose, glucosamine and galactose, however, were approximately 1:1:1 for all four lymphocyte preparations.Bursectomy or thymectomy in combination with whole body irradiation altered the carbohydrate composition significantly.The results suggest the possibility that the carbohydrate composition can be used as a marker for different lymphocyte populations. The results are also discussed in respect to the hypothesis that carbohydrate determinants on the cell surface determine the migration of lymphocytes.