bluuebunny/crossref_metadata_2025_split huggingface dataset:
Precursor Forms of the Subunits of Nitrate Reductase inchlAandchlBMutants ofEscherichia coliK12
Authors: GIORDANO Gérard , GRILLET Lydia , POMMIER Janine , TERRIERE Claude , HADDOCK Bruce A. , AZOULAY Edgard 
Published: March 2005

The synthesis of nitrate reductase by a parentalEscherichia coliK12 strain and its isogenicchlAandchlBmutants has been analyzed by protein double labelling with L‐[4,5‐3H]leucine and sulphur‐35 and by immunoprecipitation using specific antiserum. ThechlAandchlBmutants although defective in nitrate reductase activity retain the ability to synthesise the different polypeptides that are normally required for functional enzyme activity. In addition the data shows the following.These polypeptides are present in unequal quantities in the membrane and in the cytoplasm of the cells. ThechlBmutant synthesizes three times more nitrate reductase than thechlAmutant.The subunit composition of the membrane‐bound nitrate reductase present in the two mutants is different.Membrane preparations from thechlBmutant contain the three subunits α, β, γ in a ratio which is similar to the wild type.In thechlAmutant the two subunits β and γ are missing and the level of α subunit is very low. In the same membrane a 48000‐Mrsubunit (polypeptide β′) precipitable by nitrate reductase antiserum has been found. ThechlAandchlBmutants accumulate the three subunits α,β, and γ in different proportion and concentrations in the cytoplasm unlike the parental strain.The cytoplasm from thechlAmutant also contains the β′ polypeptide found in the membrane fraction of this mutant and in addition contain another polypeptide designated α′ of molecular weight 105000 which is precipitated by the nitrate reductase antiserum.The formation of particulate active nitrate reductase can be achieved by mixing the supernatant fractions of thechlAandchlBmutants (complementation)and procedes by two distinct but mutually dependent stages. Following reconstitution of activity the two peptides α′ and β′ present in the supernatant fraction of thechlAmutant, disappear. Analysis of the immunoprecipitate polypeptides present in both the soluble and particulate nitrate reductase protein after reconstitution suggests that these polypeptides are precursors of the α and β subunits following a process that remains to be elucidated.
Bromopyruvate as an Affinity Label for Baker's Yeast Flavocytochrome b2
Authors: ALLIEL Patrick M. , MULET Christine , LEDERER Florence 
Published: March 2005

We have reported in a previous communication a kinetic study showing bromopyruvate to behave as an active‐site‐directed reagent for flavocytochrome b2. It is shown here that inactivation is accompanied by incorporation of 3 mol reagent/subunit of oxidized intact enzyme and 4 mol reagent/ subunit nicked enzyme. Only one of the modifications is presumed to be responsible for activity loss. All labeled groups are found to be cysteines.Incubation of reduced nicked enzyme with bromopyruvate results in total protection of activity and loss of only one sulfhydryl group. A subsequent incubation in the presence of the competitive inhibitor sulfite leads to some more loss of non‐essential groups. After these two pretreatments, incubation in the presence of bromo[2‐14C]pyruvate results in incorporation of 1.2‐1.5 mol reagent/ subunit concomitant with the loss of about 0.8 active site.A study of the distribution of label between fragments α and β has been carried out using gel electrophoresis and Sephadex filtration after selective proteolysis. It is shown that the active‐site sulfhydryl group corresponds to one of the four cysteines situated in the last two thirds of fragment α.The structural and functional implications of these results is discussed
Methionyl‐Transfer‐RNA Transformylase from Escherichia coli
Authors: KAHN Daniel , FROMANT Michel , FAYAT Guy , DESSEN Philippe , BLANQUET Sylvain 
Published: March 2005

A large‐scale purification procedure of methionyl‐tRNA transformylase (10‐formyltetrahydro‐folate:l‐methionyl‐tRNA N‐formyltransferase) from Escherichia coli is described. The purification (10000‐fold) produces 22 mg homogeneous enzyme from 5 kg wet cells with a yield of 45%. The specific activity of the enzyme in the reaction of formylation of methionyl‐tRNAMetf is 30‐fold higher than that reported by others [Dickerman, H. W., Steers, E., Jr, Redfield, B. G. and Weissbach, H. (1967) Biol. Chem. 242, 1522‐1525].The enzyme is a monomer of molecular weight 32000, based on high‐speed equilibrium sedimentation, small‐angle neutron scattering, molecular sieving on Sephadex and gel electrophoresis in the presence of sodium dodecyl sulfate. Its absorption coefficient has been determined as A280nm=1.4 ± 0. 1 cm2. mg−1.The Michaelian parameters of the transformylation reaction have been measured for each substrate at 25 °C, pH 7. 6, in the presence of 150 mM KC1 and 7 mM MgCl2. Km values are 13.5 ± 1.5 μ and 0.35 ± 0.05 μ for 10‐formyltetrahydrofolate and for methionyl‐tRNAMetf respectively. A V value of 20 ± 2 s−1 is found at saturation of both substrates.The tryptophan fluorescence of transformylase is remarkably sensitive to the bindings of 10‐formyltetrahydrofolate and tRNAMetf This property enables one to determine 1:1 stoichiometries of the enzyme with either 10‐formyltetrahydrofolate, tRNAMetf at high ionic strength. Stability of the tRNAMetf transformylase complex is improved upon aminoacylation of tRNA. The observed association constant for the interaction between enzyme and tRNAMetf is sensitive to the concentration of cations (K+ or/and Mg2+). This behaviour has allowed us to estimate to 4.6 ± 0.7 as the number of ion pairs formed between tRNAMetf and transformylase.
The Subunit Composition of Mammalian Cytochrome c Oxidase
Authors: MERLE Peter , KADENBACH Bernhard 
Published: March 2005

Cytochrome c oxidase from rat liver mitochondria was separated into 12 different protein subunits by application of a highly resolving sodium dodecylsulfate/gel electrophoretic system of different compositions. The 12 protein subunits are shown to represent integral components of mammalian type cytochrome c oxidase for the following reasons.

All 12 subunits copurify through various purification procedures.
The subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one.
All 12 subunits are present in the complex at one to one stoichiometric amounts.
A similar composition of 12 subunits was also found for cytochrome c oxidase from rat kidney, pig heart, rabbit liver and stone‐marten liver.The difference between our results and all other published data on the subunit composition of mammalian‐type cytochrome c oxidase, based on gel electrophoretic analysis, is due to the insufficient resolving power of previously used gel systems and the very similar molecular weight of subunits VIa,b,c and VIIa,b,c.
The Topography of the 5′ End of 16‐S RNA in the Presence and Absence of Ribosomal Proteins S4 and S20
Authors: EHRESMANN Chantal , STIEGLER Patrick , CARBON Philippe , UNGEWICKELL Ernst , GARRETT Roger A. 
Published: March 2005

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16‐S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16‐S RNA.The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease‐resistant S4 RNA prepared from renatured 16‐S RNA in the absence of ribosomal protein.These data yield important information on the topography and organisation of the 5′ third of the 16‐S RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16‐S RNA.
The Stability of Ferricytochrome c
Authors: MOORE Geoffrey R. , WILLIAMS Robert J. P. 
Published: March 2005

The temperature dependence of the nuclear magnetic resonance spectrum of horse ferricytochrome c is described. The protein maintains an ordered structure over the temperature range 20 °C to 77 °C.The temperature dependence of the spectrum of ferricytochrome c arises from a number of causes including the paramagnetism of the ferric ion and protein structural changes. Preliminary analysis of the data show that the region of the protein about Ile‐57 is flexible. Comparison of the data with the analogous data for horse ferrocytochrome c reveals that there is a small difference in structure between cytochrome c in its two oxidation states in the region about Ile‐57.
Multiplicity of Cytochrome P‐450 in Primary Fetal Hepatocytes in Culture
Authors: KREMERS Pierre , GOUJON Francine , DE GRAEVE Jean , VAN CANTFORT Jacques , GIELEN Jacques E. 
Published: March 2005

Primary fetal rat hepatocytes in culture display different monooxygenase activities which can be induced by several chemical inducers. Until now, these hepatocytes were believed to produce only one single cytochrome P‐450 species, namely the cytochrome P1‐450 (or P‐448). It now seems possible to induce other cytochrome P‐450 species in these hepatocytes, providing that they receive an appropriate hormonal treatment.We have examined the effect of dexamethasone on the cytochrome P‐450 type supporting different enzymic activities (aryl hydrocarbon hydroxylase, ethoxycoumarin deethylase, aldrin monooxygenase). Our results show that the presence of dexamethasone in the culture medium produces qualitative and quantitative changes in the monooxygenase‐supporting cytochrome(s) P‐450. For low dexamethasone concentrations (between 1 nM and 0.1 μM) a cytochrome P‐450 is formed displaying biochemical and biophysical properties similar to those induced by phenobarbital in the adult rat liver. At higher concentrations (above 1 μM), similar qualitative changes are observed; but a quantitative phenomenon occurs, the (cytochrome P‐450)‐dependent enzymic activities being also induced. Dexamethasone also has a synergistic effect on the induction of enzymic activity by the mixture of phenobarbital plus benzanthracene.The various biochemical changes induced by dexamethasone in the fetal cell cultures parallel those observed in vivo during the perinatal period of life. The cell culture system may thus constitute an interesting model for studying the ontogenic development of liver monooxygenases.
The First Step in the Polymerisation of Actin
Authors: ROUAYRENC Jean‐François , TRAVERS Franck 
Published: March 2005

In the presence of certain cations (e.g. K+ or Mg2+) actin polymerizes. Below a certain concentration (the critical concentration) the monomer G‐actin does not polymerize on the addition of K+ or Mg2+. However, the proteolysis experiments of Rich and Estes [J. Mol. Biol. 104, 777–792 (1976)] strongly suggest that cations induce a change in conformation of G‐actin leading to a novel form of actin, G*‐actin. This conformational change may be the first step in the polymerization of actin.We have studied G*‐actin induced by K+, by difference spectroscopy. We show that G*‐actin is a monomer and we confirm that the bound ATP is not cleaved.We also studied the G‐actin—G*‐actin equilibrium at 4 °C as a function of K+ or Mg2+ concentration. With KCl, the transformation can be accounted for as a screening effect. The effect of Mg2+, is more specific and the chance in conformation of the G‐actin could result from the binding of two or three Mg2+ ions/molecule. We suggest that the G‐actin—G*‐actin transformation results from the neutralization of a polyanionic region on the actin surface and that this region could be the highly negatively charged N terminus.
The Binding of Dansylated Initiation Factor 3 to the 30‐S Subunit of Escherichia coli: A Fluorimetric and Biochemical Study
Authors: BOX Rainer , WOOLLEY Paul , PON Cynthia 
Published: March 2005

Initiation factor 3 (IF‐3) has been labelled using dansyl (1‐dimethylaminonaphthalene‐5‐sulphonyl) chloride under conditions designed to preserve the biological activity of the factor. The sites of modification of the IF‐3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions. However, if an IF‐3 molecule bears more than one dansyl group on average then its activity is lost. The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction. Spectrofluorimetric analysis of the dansyl‐IF‐3 leads to the following conclusions. (a) The motion of the dansyl label does not change greatly upon binding to the 30‐S subunit. (b) The label is not close enough to any tryptophan group of the ribosorne in the 30‐S‐subunit · IF‐3 complex to allow energy transfer. (c) The IF‐3 chain is folded so as to bring the tyrosine groups close to the dansyl‐binding sites. (d) The stoichiometry of the binding of IF‐3 to 30‐S ribosomal subunits is close to 1:1 and the binding constant is 2 × 107 M−1.IF‐3 also binds non‐covalently the fluorescent indicator 8‐anilinonaphthalene 1‐sulphonate (ANS) with an apparent binding constant of approximately 8000 M−1. An interaction between ANS and poly(A‐U‐G), both bound to IF‐3. was demonstrated. Combining these results with those for dansyl‐IF‐3 leads to a model for the interaction between IF‐3 and the 30‐S subunit involving a combination of ‘hydrophobic’ and electrostatic attraction between the factor and ribosomal RNA.
N‐Glycosylation of Yeast Proteins
Authors: SHARMA Chandra B. , LEHLE Ludwig , TANNER Widmar 
Published: March 2005

The enzyme transferring the oligosaccharide from DolPP‐(GlcNAc)2(Man)9(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from DolPP‐[14C]saccharidcs to the hexapeptide Tyr‐Asn‐Leu‐Thr‐Ser‐Val. The rate of transfer was linear for 20 min, with about 40% of dolichyl‐diphosphate‐bound radioactivity transferred to the peptide.The solubilized enzyme has been characterized as follows:

The enzyme is most efficiently solubilized (60% of the membrane‐associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1%.
Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10 mM yields only one third the activity observed with Mn2‐.
The enzyme transfers besides dolichyl‐diphosphate‐bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)1 and (GlcNAc)2; the rate decreases in this order. No transfer is observed from Dol PP‐(GlcNAc)2(Man)9 and from DOlPP‐GlcNAc.
The Km value for DolPP‐(GlcNAc)2(Man)9(Glc)3 of 0.5 μM does not differ significantly from that for DolPP‐(GlcNAc)2 of 1.2 μM. A broad pH‐optimum for the reaction with both substrates was found between 6.5 and 7.7.
However, a clear difference in Km values for the hexapeptide was observed with different dolichol‐linked sugar derivatives. With DOlPP‐(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half‐maximal transfer rate, whereas 0.05 mM peptide were sufficient with (GlcNAc)2(Man)9(Glc)3 as donor.
General Shape and Hapten‐Induced Conformational Changes of Pig Antibody against Dinitrophenyl
Authors: CSER László , FRANĚK František , GLADKIKH Irina A. , KUNCHENKO Alexandr B. , OSTANEVICH Yuriy M. 
Published: March 2005

Pig antibodies against dinitrophenyl were studied by neutron small‐angle scattering and X‐ray small‐angle scattering with particular attention to the analysis of cross‐section plots and determination of the radii of gyration of cross‐section. The experimentally determined molecular parameters Rg (radius of gyration), Rq1 and Rq2 (two different radii of gyration of cross‐section characterizing every antibody sample) show that the shapes of the two antibody types, precipitating and non‐precipitating, are similar. The non‐precipitating antibody is slightly more compact.The parameters Rg, Rq1 and Rq2 of complexes of antibodies with the hapten, 8‐dinitrophenyl‐5,8‐aza‐4‐oxooctanoic acid, are smaller than those of the free antibody. This indicates that a conformational change is induced by the binding of the hapten. The character of the change of parameters is consistent with a view that the observed contraction of the molecule proceeds via similarity transformation.In order to design a model of a pig antibody molecule, isolated building blocks of the molecule, the Fab and Fc fragments, were first studied. A comparison of the scattering curves with various models of fragments showed, however, that the isolated fragments acquire in solution elongated rod‐like shapes.Over 300 tentative models of the intact antibody molecule, built of small identical spheres, were constructed before a good fit with the experimental data was achieved. The most probable models have a cavity in the Fc part and the Fab parts are either fully extended or slightly bent downwards to the Fc part.
Effect of Ammonium Ion on Pyrimidine Synthesis de novo in Isolated Rat Hepatocytes
Authors: BARTON Peter A. , HOOGENRAAD Nicholas J. 
Published: March 2005

Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides, orotic acid and the acid‐hydrolyzed product of carbamoylaspartic acid by ion‐exchange chromatography and high‐pressure liquid chromatography show a marked stimulation in the incorporation of [14C]bicarbonate in incubations with added ammonium ions. The incorporation into total uridine nucleotides (ΣUMP) was increased twofold in the presence of 5 mM ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling of carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyrimidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines, there was no increase in the hepatocyte content of ΣUMP, which was 11.5 μmol/g dry weight, although the orotic acid content increased from 0.09 μmol/g dry weight in the absence of added ammonium ions (but in the prcsence of 2 mM l‐glutamine) to 8.6 μmol/g dry weight with 5 mM ammonium ion.The stimulated incorporation of [14C)bicarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80% of label in the uracil ring was present at position C‐2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5% foetal calf serum and 20 mM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium ion. The stimulatory effect of ammonium ions on incorporation of bicarbonate into pyrimidines was almost completely reversed by 5 mM l‐ornithine and was partially reversed by 1 mM l‐ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed.
Acetylcholine Receptor from Mammalian Skeletal Muscle
Authors: SHORR Robert G. , LYDDIATT Andrew , LO Mathew M. S. , DOLLY J. Oliver , BARNARD Eric A. 
Published: March 2005

The acetylcholine receptor of cat denervated skeletal muscle was solubilised with Triton X‐100 in the presence of protease inhibitors and was shown to have a sedimentation coefficient of about 9 S. This oligomer can be converted to a smaller, active 4‐S species.
This 9‐S glycoprotein was purified to homogeneity (showing pI = 5.0) by improved biospecific chromatography on a‐neurotoxin and lectin affinity gels, and shown to bind specifically 10–11.5 μmol [2,3‐3H]propionyl‐α‐bungarotoxin/g protein. The association rate constant (3 × 105 M−1 s−1 at 25°C) for this reaction was similar to that observed with membrane‐bound or unpurified receptor; affinity constants for nicotinic ligands were also similar in all these cases.
By a variety of techniques, a major polypeptide of Mr about 43000 was detected in the pure protein. Likewise, both 9‐S and 4‐S oligomers isolated it a pure state at high yield (∼ 80%) by a novel technique using anti‐toxin immunoglobulin, contained the same size of subunit.
Sub‐synaptic and extra‐synaptic forms of the receptor were alkylated specifically in the membrane‐bound state with the affinity reagent bromo[3H]acetylcholine. As in the case of the pure receptor from denervated muscle, the same size polypeptide (Mr 43000) was labelled. This was true, also, for both the 9‐S and the 4‐S oligomer of the denervated muscle receptor.
Proposed oligomeric structures of acetylcholine receptors containing single and multiple‐size subunits are discussed.
Molecular Forms of the Acetylcholine Receptor from Vertebrate Muscles and Torpedo Electric Organ
Authors: LO Mathew M. S. , DOLLY J. Oliver , BARNARD Eric A. 
Published: March 2005

Multiple forms of the acetylcholine receptor solubilised from cat denervated muscle were separated by velocity sedimentation centrifugation. The kinetic properties of the two main forms (with sedimentation coefficients of 9 S and 4 S) were investigated using a pure preparation of a suitable probe, [3H]propionyl‐α‐bungarotoxin. The binding of this toxin to each of these forms of the muscle receptor was consistent with a simple bimolecular reaction with a homogeneous class of binding sites. Negligible dissociation of the receptor‐toxin complex was observed. This behaviour was also found for the different forms of the acetylcholine receptor of chick embryo muscle and of Torpedo marmorata electric organ. Association rate constants for binding of the 3H‐labelled a‐toxin to receptor from chick embryo muscle and the 9‐S and 4‐S forms from cat denervated muscle were 0.54 × 105, 1.76 × 105 and 2.69 × 105 M−1 s−1 respectively, at 25°C. The values obtained for the 9‐S and 13‐S forms of receptor from T. marmorata were 4.51 × 105 and 9.93 × 105 M−1 s−1 respectively.The reaction of the 3H‐labelled α‐toxin with the receptor was second‐order and linear in the presence of an antagonist, as in its absence, for the 4‐S and 9‐S forms of the cat denervated muscle receptor. This reaction of the receptor was inhibited by cholinergic ligands, with Ki values for two antagonists tested being greater with the 4‐S form than with the 9‐S form. Apparent negative interaction is observed for antagonists with this receptor, with Hill coefficients of about 0.78 and 0.64 for the 4‐S and 9‐S forms respectively. A ligand‐induced affinity increase, produced by the agonists but not by the antagonists, was observed in this reaction for both forms of the muscle receptor. Two agonists tested showed no difference between these forms in their highaffinity states in either their binding affinities or HIII coefficients.