bluuebunny/crossref_metadata_2025 huggingface dataset:
Cobra Venom Acetylcholinesterase
Authors: RABA Raivo , AAVIKSAAR Aavo , RABA Mari , SIIGUR Jüri
Published: March 2005

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate.

Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67000 ± 2000. At high enzyme concentrations (> 0.2 mg/ml, > 1 μM) and ionic strength 0.1 M, it reversibly tends to form higher‐molecular‐weight 7.1‐S aggregates.

Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.
Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein
Authors: STANELONI Roberto J. , UGALDE Rodolfo A. , LELOIR Luis F.
Published: March 2005

The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose. Paper chromatography allowed the separation of oligosaccharides (obtained by acid hydrolysis of the dolichyl diphosphate derivative) containing 1, 2 and 3 glucose residues. Using this procedure it was found that the addition of all three glucoses to the dolichyl diphosphate oligosaccharide occur with dolichyl phosphate glucose as donor. Furthermore only the compound with three glucoses was used as donor in the transfer to protein. The addition of glucose to exogenous dolichyldiphosphate oligosaccharide labelled by transfer from radioactive guanosine diphosphate mannose was detected.
Pulse‐Fluorometry Study on Actin and Heavy Meromyosin Using F‐Actin Labelled with N‐(1‐Pyrene)maleimide
Authors: KOUYAMA Tsutomu , MIHASHI Koshin
Published: March 2005

The single‐photoelectron counting technique was used for measurement of the fluorescence decay kinetics of N‐(1‐pyrene)maleimide conjugated to the fast reactive cysteine of actin. The fluorescence decay curve of the labelled G‐actin could not be described by a single‐exponential function but by a double‐exponential function. Polymerization of actin was accompanied by significant changes in the decay parameters of the two decay components. We found that the ionic strength, which plays an important role in the G‐F equilibrium, scarcely affected these parameters provided that the labelled actin exists in the monomeric state. Thus it is suggested that the conformational change of actin protomer occurs at the time of association. When heavy meromyosin was added to the labelled F‐actin, the decay parameter changed monotonically on increasing saturation of binding of heavy meromyosin and it levelled‐off around a ratio of heavy meromyosin:actin of 0.5 mol/mol. Decay parameters under the influence of heavy meromyosin had values intermediate between those observed for the labelled G‐actin and for the labelled F‐actin. Therefore, it is suggested that binding of heavy meromyosin to F‐actin alters the conformation of actin protomer towards that similar to G‐actin.
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
Purification and Physicochemical Properties of Ricins and Agglutinins from Ricinus communis
Authors: Thomas T.‐S. , LI Steven S.‐L.
Published: March 2005

Two toxin ricins and two agglutinins have been purified from the seeds of Ricinus communis by an improved procedure based on ion‐exchange chromatography and gel filtration. One of the two purified ricins binds to Sepharose 4B while the other does not. The physicochemical properties of the four Ricinus lectins are presented and the possible relationships of these Ricinus lectins to those previously described are discussed.
Synthesis of 5′ Fragments of Formylmethionine Transfer Ribonucleic Acid and Their Reconstitution with a Natural Three‐Quarter Molecule
Authors: OHTSUKA Eiko , NISHIKAWA Satoshi , FUKUMOTO Ryoichi , UEMURA Haruki , TANAKA Toshiki , NAKAGAWA Eiko , MIYAKE Tetsuo , IKEHARA Morio
Published: March 2005

An eicosanucleotide C–G–C–G–G–G–G–U–G–G–A–G–C–A–G–C–C–U–G–Gp corresponding to the bases 1–20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C–G–C–G, G–G–G–U–G–G and A–G–C–A–G–C–C–U–G–Gp using RNA ligase from T4‐infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [γ−32P] ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3′‐three‐quarter molecule obtained by limited digestion with RNase Tl of the natural tRNA fMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl‐tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.
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.
Active Centers of α‐Chymotrypsin and of Streptomyces griseus Proteases 1 and 3
Authors: BAUER Carl‐Axel
Published: March 2005

A number of peptides of the general formula Ac‐Pro‐Ala‐X‐Phe‐NH2, where X = Gly, Ala, Leu, Phe or Pro, have been synthesized for the study of S2‐P2 enzyme‐substrate interactions in three serine proteases: α‐chymotrypsin and Streptomyces griseus proteases 1 and 3. All three enzymes interacted favorably with those peptides with hydrophobic, non‐aromatic, P2 amino acid residues, leucine being optimal, The increase in kcat/km on going from the P2 glycine to the P2leucine peptide was 30‐fold in α‐chymotrypsin and greater than 150‐fold and 350‐fold in the Streptomyces enzymes, being due to both increased affinities and higher acylation rates. Hydrophobic S2‐P2 interactions, particularly in S. griseus proteases 1 and 3, appear to be the most important enzymesubstrate interactions apart from S1‐P1. The kinetic data is discussed in relation to the tertiary structures of the active centers of the enzymes.
Trans‐membrane Translocation of Proteins
Authors: von HEIJNE Gunnar
Published: March 2005

A detailed, computerized procedure for analysing the translocation process of any protein with known sequence from a physico‐chemical point of view is presented and used to gain a better understanding of the molecular ‘rules’ that govern the final outcome of the process. With the aid of this procedure a number of testable predictions of the orientations of particular membrane‐bound proteins can be made. It is also suggested that ovalbumin, the only known secreted protein lacking a cleavable prepiece, initiates translocation in a way that differs from other secreted proteins.
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.
Nuclear‐Magnetic‐Resonance Studies of Eukaryotic Cytochrome c
Authors: MOORE Geoffrey R. , WILLIAMS Robert J. P.
Published: March 2005

The aromatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double‐resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X‐ray structure of cytochrome c. 33 resonances arising from 39 aromatic protons of ferrocytochrome c, and 18 resonances arising from 27 aromatic protons of ferricytochrome c have been assigned.
Nuclear‐Magnetic‐Resonance Studies of Eukaryotic Cytochrome c
Authors: MOORE Geoffrey R. , WILLIAMS Robert J. P.
Published: March 2005

The aliphatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double‐resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X‐ray structure of cytochrome c. There are eight firmly assigned methyl resonances of ferrocytochrome c and seven firmly assigned methyl resonances of ferricytochrome c.
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.
Comparison of the Structures of Various Eukaryotic Ferricytochromes c and Ferrocytochromes and Their Antigenic Differences
Authors: MOORE Geoffre R. , WILLIAMS Robert J. P.
Published: March 2005

The nuclear magnetic resonance spectra of various eukaryotic ferricytochromes c and ferrocytochromes c are described. The proteins from the species donkey, cow, dog, rabbit, chicken and pigeon were investigated. The conformations of these proteins detected by NMR were compared to those of horse and tuna cytochromes c and in some cases small differences were found. These differences in structure were shown to correlate with antigenic differences between the various proteins.
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.
31P Nuclear Magnetic Resonance Studies of Energy Transduction in Rhodopseudomonas sphaeroides
Authors: NICOLAY Klaas , KAPTEIN Robert , HELLINGWERF Klaas J. , KONINGS Wil N.
Published: March 2005

31P nuclear magnetic resonance spectra of the phototrophic bacterium Rhodopseudomonas sphaeroides reveal the presence of inorganic phosphate, sugar phosphates and two non‐identified P,P1‐diesterified pyrophosphate compounds. Due to the presence of paramagnetic cations the resonances of these compounds can only be detected after repeated washing of the bacterial cells with a buffer, containing EDTA plus excess Mg2+.

Washing with Mg2+‐free EDTA buffer deteriorates the structural integrity of the membranes of Rps. sphaeroides. This is indicated by the appearance of an extra resonance peak in the spectra of these cells in a region where the phosphoiipids absorb and by a fivefold increase in proton permeability of the cytoplasmic membrane of Rps. sphaeroides under these conditions. Upon illumination of the cell suspension in the NMR tube the generation of a transmembrane pH gradient can be inferred from the shift in the resonances of extracellular and intracellular inorganic phosphate. Intracellular inorganic phosphate shows one homogeneous resonance peak upon illumination. This demonstrates that the mixing system, which has been developed for this application, functions efficiently. The magnitude of the light‐dependent pH difference is 0.8 at an external pH 6.

The width at half height of the internal inorganic phosphate peak is essentially independent of internal pH from pH 5–8, remains unchanged upon addition of uncoupler and is inversely proportional to the number of EDTA washings applied. These observations indicate that the inorganic phosphate NMR peak width is predominantly determined by the presence of a residual amount of paramagnetic cations, rather than by a broad distribution of internal pH values over the cells.

Ionophores have an effect on the light‐dependent pH‐gradient in accordance with the chemiosmotic theory: valinomycin increases, and carbonylcyanide p‐trifluoromethoxyphenylhydrazone decreases, the magnitude of this gradient.
Conformational Changes and Possible Structure of the Oxoglutarate Translocator of Rat‐Heart Mitochondria Revealed by the Kinetic Study of Malate and Oxoglutarate Uptake
Authors: SLUSE‐GOFFART Claudine M. , SLUSE Francis E. , DUYCKAERTS Claire , RICHARD Marc , HENGESCH Paul , LIÉBECQ Claude , Bertrand Laurette , Dethier Eli , Cuvelier‐Klimek Monique , Salmon José
Published: March 2005

Initial rates of the exchanges [14C]malateout:malatein, ([14C]oxoglutarate + malate)out: malatein and ([14C]malate + oxoglutarate)out: malatein catalysed by the oxoglutarate carrier of rat‐heart mitochondria have been studied under conditions where internal and external substrates may be varied.

It is shown that contrary to external oxoglutarate which induces a conformational change of the translocator subunit to which it binds, external malate does not induce conformational changes during its binding and is a Michaelian substrate.

The study of the effect of external malate on the rate of oxoglutarate uptake shows that external malate and external oxoglutarate are competitive.

External oxoglutarate affects the catalytic rate constant of malate uptake in a modulated way.

After substrate binding, the exchange reaction between an external dicarboxylate and an internal dicarboxylate is accompanied by conformational changes. The particular form of the rate equation strongly suggests that during a first step the external substrate bound to an external binding subunit at the external surface of the membrane, and the internal substrate bound to an internal binding subunit at the internal surface of the membrane, are transferred to a catalytic subunit (channel?) deeper in the membrane. Two models, one with a single channel, and the other with several associated channels, are proposed.

It is demonstrated that a binding subunit which has transferred its substrate to a catalytic subunit is left in a conformation which does not depend on the substrate that has ‘passed through it’. It is also demonstrated that all the catalytic subunits are identical. These theoretical deductions allow a simple description of the complicated effect that external oxoglutarate has on the rate of malate uptake.

The fact that all the external binding subunits are equivalent regarding external malate binding and that all the catalytic subunits are identical support the view that the mitochondrial preparation contains a single species of oxoglutarate translocator and not an isozymic mixture.
Cell‐Free Synthesis of Hemocyanin from the Scorpion Androctonus australis
Authors: ALLIEL Patrick M. , DAUTIGNY André , LAMY Josette , LAMY Jean‐N. , JOLLéS Pierre
Published: March 2005

Translation of Androctonus australis poly(A)‐RNA in vitro led to a number of polypeptides products (8–10) of 70–73 kDa analyzed by two‐dimensional gel electrophoresis and identified by immunoprecipitation with an anti‐ (dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico‐chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross‐reacting subunits. Each polypeptide chain is coded by a different messenger without significant post‐translational events. Hemocyanin could be detected among the translation products of the poly(A)‐RNA isolated from the cuticle under the carapace.
Calcium and Cadmium Binding to Troponin C
Authors: TELEMAN Olle , DRAKENBERG Torbjörn , FORSÉN Sture , THULIN Eva
Published: March 2005

Proton NMR is used to compare the structural changes induced in bovine cardiac troponin C on binding of cadmium and calcium ions. The same spectral changes are observed for both ion species. The rate of the conformational changes associated with cadmium binding to the two high‐affinity sites is slow, that associated with cadmium ions binding to the low‐affinity site is high.

113Cd‐NMR spectra of cardiac troponin C feature two signals interpreted as due to cadmium ions bound to the strong sites.

Strong arguments are given in favour of cooperativity in binding of the first two cadmium or calcium ions to cardiac and skeletal muscle troponin C.
A113Cd and 1H NMR Study of the Interaction of Calmodulin with D600, Trifluoperazine and Some Other Hydrophobic Drugs
Authors: ANDERSSON Anita , DRAKENBERG Torbjörn , THULIN Eva , FORSÉN Sture
Published: March 2005

The interaction of calmodulin with D600 (a methoxy derivative of verapamil), trifluoperazine and some other drugs was studied by 113Cd and 1H NMR. All four cation binding sites of calmodulin were found to be affected by the binding of the drugs to calmodulin. The physiologically active and inactive forms of felodipine were found to give qualitatively the same changes in the 113Cd NMR spectra of calmodulin. The interpretation of this observation in terms of the physiological relevance of the binding to calmodulin is discussed. The binding constants for the two strongly bound trifluoperazine molecules were found to differ by one or two orders of magnitude.
Cis‐Platinum Induced Distortions in DNA
Authors: den HARTOG Jeroen H. J. , ALTONA Cornelis , van BOOM Jacques H. , MARCELIS Antonius T. M. , van der MAREL Gijs A. , RINKEL Lambertus J. , WILLE‐HAZELEGER Gerry , REEDIJK Jan
Published: March 2005

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G‐G chelated deoxytrinucleosidediphosphate platinum complex cis‐Pt(NH3)2{d(GpCpG)}, on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis‐Pt(NH3)2{d(Gp)}2, cis‐Pt(NH3)2{d(pG)}2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis‐Pt(NH3)2]2+.

Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring. This feature is also apparent in ribose mononucleotides and is possibly caused by an increased anomeric effect. In cis‐Pt(NH3)2{d(pG)}2 the phase angle of pseudorotation of the S‐type sugar ring is 20° higher than in ‘free’ d(pG) which might be an indication for an ionic interaction between the positive platinum and the negatively charged phosphate.

It appears that d(GpCpG) reverts from a predominantly random coil to a normal right‐handed B‐DNA‐like single‐helical structure at lower temperatures, whereas the conformational features of cis‐Pt(NH3)2{d(GpCpG)} are largely temperature‐independent. In the latter compound much conformational freedom along the backbone angles is seen. The cytosine protons and deoxyribose protons exhibit almost no shielding effect as should normally be exerted by the guanine bases in stacking positions. This is interpreted in terms of a ‘turning away’ of the cytosine residue from both chelating guanines. Conformational features of cis‐Pt(NH3)2{d(GpCpG)} are compared with the ‘bulge‐out’ of the ribose‐trinucleotide m62ApUpm62A.
Cell‐Wall Lipopolysaccharide of Escherichia coli O114:H2
Authors: DMITRIEV Boris A. , LVOV Vjacheslav , TOCHTAMYSHEVA Natalya V. , SHASHKOV Alexandr S. , KOCHETKOV Nicolay K. , JANN Barbara , JANN Klaus
Published: March 2005

The O‐specific polysaccharide of the 0114 antigen (lipopolysaccharide) of Escherichia coli 0114 and oligosaccharides obtained from it by Smith degradation and hydrogen fluoride solvolysis were analyzed, using proton and 13C nuclear magnetic resonance spectroscopy and methylation. The results indicated that the 0114 polysaccharide has the tetrasaccharide repeating unit α‐N‐acetylglucosamine(1 → 4)β‐3,6‐dideoxy‐3‐(N‐acetyl‐L‐seryl)aminoglucose(1 → 3)β‐ribofuranose(1 → 4)galactose. In the polysaccharide the repeating units are joined through β1 → 3‐galactosyl linkages. This structure is compared with that of the serologically cross‐reacting Shigella boydii 08 antigen and the serological similarity is discussed.
Effect of pH and Monovalent Cations on the Ionization State of Phosphatidylglycerol in Monolayers
Authors: LAKHDAR‐GHAZAL Faouzi , TICHADOU Jean‐Louis , TOCANNE Jean‐François
Published: March 2005

Ionization of the acidic phospolipid phosphatidylglycerol has been studied by measuring the surface potential of monomolecular films of the lipid as a function of the aqueous subphase pH and the concentration of monovalent cations (Li, Na, Cs). It is shown that the experimental data can be interpreted by means of the Gouy‐Chapman theory in its simplest formulation, provided an adsorption of cations at the membrane surface is accounted for. This allows us to predict the ionization state of the lipid for given ionic conditions in the subphase. Above pH 4, for subphase ion concentration higher than 10 mM, or for ion concentrations above 0.1 mM at pH 5.6, phosphatidylglycerol is fully deprotonated. Within the limits of our theoretical approach, association constants of the cations to the lipid lie around 0.1–0.6 M−1.
Characterisation of Hepatocyte Sub‐populations Generated by Centrifugal Elutriation
Authors: SUMNER Ian Gordon , FREEDMAN Robert Ben , LODOLA Alberto
Published: March 2005

We describe protocols for the fractionation of isolated hepatocytes into eight sub‐populations using centrifugal elutriation. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre‐perfused with one of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P‐450 content and response to induction by 3‐methylcholanthrene and phenobarbitone; the distribution of lactate dehydrogenase, glucose‐6‐phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the sub‐populations is also reported. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of eight factors studied, the distributions of six were consistent with the sub‐populations being derived from different acinar zones. Two major discrepancies were noted however, the distribution of pyruvate kinase activity and the response of the sub‐populations to phenobarbitone.

We conclude from this study that while a metabolic heterogeneity was revealed in the sub‐populations generated, further characterisation is required to determine whether acinar zone separation has occurred and if so to what extent.
Solvent Isotope Effects on the Reaction Catalyzed by Yeast Hexokinase
Authors: TAYLOR Kenneth B. , COOK Paul F. , CLELAND W. Wallace
Published: March 2005

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5–1.7 for all three parameters. When 1,5‐anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5‐anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH‐dependent change in the E · glucose complex.
Developmental and evolutionary comparisons of proteins from purified ribosomal subunits in two silkmoths
Authors: KOUYANOU Sophie , FRAGOULIS Emmanuel , KAFATOS Fotis C.
Published: March 2005

Developmental comparisons of silkmoth ribosomal proteins were performed by parallel two‐dimensional electrophoretic analyses of proteins derived from developing adult wings and from ovarian follicles at the prechoriogenic and choriogenic stages of development (before and during secretion of the chorion, respectively). Proteins of 40 S and 60 S subunits were prepared and analyzed separately. A single major developmental difference was observed, exclusively in choriogenic follicles: the majority of a 40S subunit protein, S6, was shifted to a more acidic form, possibly as a result of phosphorylation. A less prominent change was apparently due to quantitative variation between two forms of one large subunit protein. The developmental comparisons were performed in two species of the genus Antheraea, with consistent results. Comparisons between the two species revealed remarkable evolutionary conservation of the ribosomal protein patterns, sharply contrasting with the evolutionary diversification of chorion structural proteins in the same two species. The only detectable interspecies differences in ribosomal components were (a) slightly more acidic behaviour of one small‐subunit protein and more basic behaviour of one large‐subunit protein in Antheraea polyphemus as compared to Antheraea pernyi, and (b) presence of an apparently species‐specific 60 S subunit protein in A. pernyi, which in A. polyphemus probably co‐migrates with a neighbouring protein.