bluuebunny/crossref_metadata_2025 huggingface dataset:
Dimerization of Deoxyribonuclease I, Lysozyme and Papain
Authors: SORRENTINO Salvatore , YAKOVLEV Gennady I. , LlBONATI Massimo
Published: March 2005

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskočil, J., and Libonati, M. (1980) Biochim. Bioplhys. Acta, 609, 40–52]. To see whether this phenomenon could be common to other enzyme‐substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease. hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg‐white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength.

Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross‐linkage with dimethyl suberimidate.

The main results of the investigation, similar to those obtained with ribonuclease A, are the following.

1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one.

2.The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme.

A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non‐specific interactions with polyelectrolytic substrates.
The Chemical Structure of Lipid A
Authors: WOLLENWEBER Horst‐Werner , BROADY Kevin W. , LUDERITZ Otto , RIETSCHEL Ernst Th
Published: March 2005

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated β1,6‐linked D‐glucosamine disaccharide molecule, carries approximately seven residues of fatty acids: one each of dodecanoic, hexadecanoic, D‐3‐hydroxytetradecanoic and D‐3‐O‐(tetradecanoyl)‐tetradecanoic acid in ester linkage and two of D‐3‐hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopoly‐ saccharide with alkali at elevated temperature leads, through a β‐elimination reaction, to the generation of amice‐ bound δ2‐tetradecenoic acid. This suggested that the 3‐hydroxyl group of amide‐bound hydroxy fatty acids carried a substituent. To elucidate the nature of the substituent, free Salmonella lipid A was methylated with methyl iodine in the presence of silver salts followed by mild acid hydrolysis, a procedure which is known to cleave amide (and not ester) bonds selectively. In the hydrolysate, by means of combined gas‐liquid chromatography/mass spectrometry the methyl esters of 3‐O‐(dodecanoyl)‐tetradecanoic and 3‐O‐(hexadecanoyl)‐tetradecanoic acid were identified. This shows that in lipid A amide‐linked 3‐hydroxytetradecanoic acid residues are 3–0‐acylated by dodecanoic and hexadecanoic acid, respectively.

Quantitative analyses suggest that the Salmonella lipid A backbone is substituted by four D‐3‐hydroxytetra‐ decanoyl residues, two being present as esters and two as amides. The nonhydroxylated fatty acids are not bound directly to the backbone. Rather, they are attached to hydroxyl groups of 3‐hydroxytet radecanoyl residues: specifically, tetradecanoic acid substitutes ester‐bound and dodecanoic and hexadecanoic acid amide‐bound 3‐hydroxytetradecanoic acid.
Carbohydrate Complexity of the Mouse Thymocyte Thy‐1 Glycoprotein as Demonstrated by Lectin Affinity and Isoelectric Focusing
Authors: CARLSSON Sven , STIGBRAND Torgny
Published: March 2005

The Thy‐I glycoprotein of mouse thymocytes was analysed with regard to the properties of its Carbohydrate part. Of the different lectins tested, partial binding of Thy‐1 from membrane extracts was found to Lens culinuris agglutinin (approximately 50%) and wheat germ agglutinin (approximately 25%). In contrast, concanavalin A bound all Thy‐I. The results indicate that there is considerable microheterogeneity in the oligosaccaride chains within the Thy‐I population.

Thy‐I was purified from thymocyte membrane extracts by concanavalin A affinity chromatography and gel filtration. The purified preparation revealed, on analysis by isoelectric focusing, at least six charge variants (isoelectric points ranging from approximately 5–9). These variants were isolated, treated with neuraminidase and tested for lectin binding and isoelectric point. It was shown that the charge heterogeneity was due to different amounts of sialic acid; even the most basic form contained at least one sialic acid residue. Furthermore, it was concluded that the degree of sialylation was not correlated to the lectin‐binding properties.

Analysis of Thy‐1 from surface 125I‐labelled thymocytes showed that the purified preparation was representative in its complexity of the Thy‐I glycoprotein exposed on the surface membrane. The possible biological implications of the findings are discussed.
Parameters Affecting the Superreactivity of the Cysteine Side Chain in Histone H3
Authors: BODE Jürgen , WINGENDER Edgar , PLANK Karin‐Heide
Published: March 2005

Tryptic digestion of the whole histone mixture from chicken erythrocytes is used to isolate the hydrophobic portion (residues 84‐115) of histone H3. The phenomenon of a superreactive thiol at Cys‐110 was reinvestigated by the reaction of a neutral, fluorogenic reagent, N‐[P‐(2‐benzimidazolyl)phenyl]maleimide, with this peptide and with the intact histone. Removing the highly basic portions of H3 leads to a strong reduction of Cys‐110 reactivities. even if a stoichiometric complex with histone H4 is reconstituted. An apparent superreactivity is found only for a non‐degraded histone H3 under conditions of low ionic strength but not for the histone complex in its native conformation. It is concluded that thiol activation is due to an artifactual cluster of positive charges around Cys‐110 rather than to a stable microenvironment.
Primary Structure of Escherichia coli Ribosomal Protein S1 and Features of Its Functional Domains
Authors: KIMURA Makoto , FOULAKI Kirani , SUBRAMANIAN Alap‐Raman , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

The complete covalent structure of ribosomal protein S1 of Eschericlziu coli has been determined and predictions made of its secondary structure. Protein S1 (E. coli MRE 600) is a single‐chain, acidic protein with 557 amino acid residues of the composition Asp43, Asn23, Thr25, Ser25, Glu60, Glnl4, Pro10, Gly48, Ala48, Val67, Met6, Ile30, Leu45, Tyr6, Phe17, His8, LYS43, Arg30, Trp7, Cys2 and an Mr, of 61159. The two ‐SH groups of S1 are located in the central region of the chain at positions 292 and 349, the latter being the reactive group whose modification results in the reported loss of the nucleic‐acid‐unfolding ability of S1, The central region also contains the majority of the tryptophan, histidine and methionine residues of S1 and is predicted to have a secondary structure dominated by β‐sheets and turns. A direct proof for the location of the nucleic‐acid‐binding domain of S1 in the central region has recently been obtained [Subramanian et al. (1981) Eur. J. Biochem. 119, 245‐249], The N‐terminal region of S1, which contains the ribosome‐binding domain has a relatively high predicted α‐helix content and no preponderance of basic amino acids. The facile trypsin‐sensitive site in S1 is located at Arg‐171, approximately at the border between the N‐terminal and central regions. The acidic and basic amino acids of S1 (32.8% of all residues) are distributed throughout the chain, often in small clusters of between two and six residues.

The amino acid sequence of S1 contains three 24‐residue stretches with strong internal homology. Two of the stretches are located in the central, RNA‐binding region, suggesting a possible role in the RNA‐binding and helix‐destabilizing functions of S1. A fragment of Mr, 104 from the central region of S1 gives an anomalously high apparent Mr, by dodecylsulfate gel electrophoresis, indicating a stable structural element therein and accounting for the apparent high Mr, of S1 as determined by gel electrophoresis.
Metal Coordination Centres of Class II Cytochromes c
Authors: MOORE Geoffrey R. , CLAYDEN Nigel .J. , WILLIAMS Robert J. P. , McCLUNE Gregory J. , ALSAADI Basim M. , ANGSTROM Jonas , AMBLFR Richard P. , VAN BEEUMEN Jozef , TEMPST Paul , BARTSCH Robert G. , MEYER Terrance E. , KAMEN Martin D.
Published: March 2005

The class II cytochromes Rhodospirillum molischianum cytochrome c', Rhodopseudomonas palustris cytochrome c556 and Agrohacterium tumefuciens (B2a) cytochrome c556 have been investigated with a variety of spectroscopic techniques. The cytochrome c' was found to be high‐spin and the two cytochromes c556 were found to be mainly low‐spin and sx‐coordinate with the fifth and sixth ligands being histidine and methionine. The implications of the different types of iron coordination are discussed.
Large‐Scale Purification of the 3'‐OH‐Terminal tRNA‐Like Sequence (n= 159) of Turnip‐Yellow‐Mosaic‐Virus RNA
Authors: FLORENTZ Catherine , BRIAND Jean‐Paul , GIEGE Richard , MENGUAL Raymond
Published: March 2005

In order to undertake structural and functional studies on the 3'‐terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl‐tRNA synthetase, we have developed large‐scale methods for purifying the tRNA‐like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA‐like fragment. Starting from 1.5 g turnip yellow mosaic virus, one obtains 400 mg RNA, which is partially digested by T1 ribonuclease and which yields 1 ‐ 2 mg pure tRNA‐like fragment after three chromatographic steps: two filtrations on Ultrogel ACA 54 and onc revcrse‐phase chromatography (RPC 5) in the presence of urea. A method has been worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous upon polyacrylamide gel electrophoresis under denaturing conditions. The isolated tRNA‐like structure can be valylated to an extent of 100% in the presence of purified yeast valyl‐tRNA synthetasc with kinetic parameters resembling those of the tRNAVal aminoacylation.
Implication of Arginyl Residues in Aminoacyl‐tRNA Binding to Ribosomes
Authors: HERNÁNDEZ Francisco , LÓPEZ‐RIVAS Abelardo , PINTOR‐TORO José Antonlo , PALACIÁN Enrique
Published: March 2005

Modification of the 50‐S subunits of Escherichia coli ribosomes with the arginine reagent phenylglyoxal produces inactivation of peptidyl transferase and inhibition of the binding of C(U)‐A‐C‐C‐A‐LeuAc, phenylalnnyltRNA and N‐acetylphenylalanyl‐tRNA to the ribosome. Hybridization experiments, using 1.25 M LiCl core particles and the corresponding split proteins from unireated and phenylglyoxal‐treated 50‐S subunits, indicate that inactivation and inhibition of binding are the effects of modification of a protein fraction, the functionality of the RNA moiety being unaffected by the reagent. The split proteins from phenylglyoxal‐modified 50‐S subunits are incorporated to 1.25 M LiCl core particles as well as those obtained from unmodified subunits, thus excluding the failure to bind as the cause of inactivation. In agreement with the general role played by the arginyl residues as positive binding sites for anionic ligands, the present results indicate that the arginyl residues or a protein fraction from 50‐S subunits might be important in the binding of aminoacyl‐tRNA and peptidyl‐tRNA to ribosomes.
Individual Assignments of the Amide Proton Resonances Involved in the Triple‐Stranded Antiparallel Pleated β‐Sheet Structure of a Long Neurotoxin, Laticauda Semifasciata I11 from Laticauda semifasciata
Authors: INAGAKI Fuyuhiko , CLAYDLN Nigel J. , WILLIAMS Robert J. P. , TAMIYA Nobuo
Published: March 2005

The chatacteristic feature of the crystal structure of erabutoxin b, a short ncurotoxin from Laticuuda semifasciata, and α‐cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple‐stranded antiparallel pleated β‐sheet structure formed by the central and the third peptide loops. In the present study, wc have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L.semifaciata using nuclear Overhauser effects (NOE) and spin‐dccoupling methods. The results show that nearly all of the slowly exchangeable amide protons are to be assigned to the back‐bone iimide protons, involved in the triple‐stranded antiparallel pleated β‐sheet structure, indicating that this sheet is stable in 2H2O solution. In contrast, the amide protons in short neurotoxins are readily exchangeable under the same experimental condition, suggesting that long neurotoxins have a more rigid sheet structure than short ones. This rigidity may come from the hydrophobic and hydrogen bond interaction between the central loop and the tail, which is not present in short neurotoxins. Since the functionally important residues are located on this β‐sheet, the different kinetic properties of the neurotoxins are well correlated with the difference in the rigidity of the β‐sheet.
Different Modes of Membrane Interactions of the Signal Sequence of Carp Preproinsulin and of the Insertion Sequence of Rabbit Cytochrome b5
Authors: BENDZKO Peter , PFEIL Wolfgang , RAPOPORT Tom A. , PREHN Siegfried
Published: March 2005

The signal segment of the secretory protein carp preproinsulin is shown to be bound by a protein receptor present in the rough endoplasmic reticulum membrane. The receptor does not bind the insertion segment of the integral membrane protein cytochrome b5. On the other hand, the insertion sequence, in contrast to the signal sequence, is dissolved into the lipid bilayer of natural and artificial membranes. Hydrophobicity or length per se of the two types of peptides cannot be responsible for their different behaviour. We rather propose that the difference resides in their tertiary structure. Insertion peptides may form a compact structure with a diffuse hydrophobic surface, presumably by internal hydrogen bonding. Signal peptides would form hydrogen bonds with a membrane‐bound receptor protein, presumably by producing a β‐sheet structure, but their extended structure in aqueous solution would not allow them to dissolve into lipid bilayers.
Light‐Induced Interactions between Rhodopsin and the GTP‐Binding Protein
Authors: BENNETT Nelly
Published: March 2005

The existence of rapid light‐induced changes of light scattering in suspensions of bovine rod outer segment membranes has been described previously [H. Kühn et al. (1981) Proc. Nutl Acud. Sci. USA, 78, 6873‐6877]. The signal observed in the presence of GTP has been interpreted as being related to the rhodopsin‐catalyzed exchange of GTP for GDP bound to the GTP‐binding protein, i.e. to the formation of the activator of the cGMP phosphodiesterase [B. K. K. Fung et al. (1981) Proc. Nutl Acad. Sci. USA, 78, 152‐ 1561. We have tested this interpretation in the present paper by investigating the relation between the light‐scattering signal and the activity of the phosphodiesterase using rapid recording techniques for both processes. All the results obtained are consistent with the above hypothesis. The amplitude of the light‐scattering signal and the activity of the phosphodiesterase are shown to present the same dependence upon the flash intensity and upon the concentration of GTP or its analog guanosine 5'‐[β,γ‐imido]triphosphate (p[NH]ppG). The results suggest that the GTP‐binding protein possesses one high‐affinity p[NH]ppG‐binding site (Kd≪ 0.1 μM). At high concentrations of GTP or p[NH]ppG the phosphodiesterase is activated in the dark and the light‐scattering signal is correspondingly reduced; both effects are prevented by previous incubation with guanosine S'‐[β‐thio]diphosphate (P[S]PG).
C1 Proteins: a Class of High‐Mobility‐Group Non‐histone Chromosomal Proteins from the Fruit Fly Ceratitis capitata
Authors: MARQUEZ Gabriel , MORAN Federico , MONTERO Francisco , FRANCO Luis
Published: March 2005

CM‐cellulose chromatography of a fraction soluble in 5% perchlorice acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which bave been purified to electrophorecal homogeneity.

C1a1, C1a2 and C1b analyse like high mobility group (HMG) non‐histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Dosophilia D1 [Rodriguez Alfageme et al. (1980) Chromosoma, 78, 1‐31] are representative of a class of insect‐specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength.

The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2.
Photochemical Inactivation of Lactoperoxidase Sensitized by Carbonyl Compounds
Authors: MÁKINEN Kauko K. , MÁKINEN Pirkko‐Liisa
Published: March 2005

2,3‐Butanedione was found to be a poor noncompetitive inhibitor (Ki∼ 0.1 M) of bovine milk lacto‐peroxidase. Irradiation of an incubation mixture of these two substances in the visible region led, however, to an irreversible loss of enzyme activity. The inactivation rates were dependent on light intensity, light wavelength and sensitizer concentration. Maximal rate of inactivation occurred at 400‐420 nm. Other dicarbonyl compounds which caused a similar photodestruction of lactoperoxidase in the visible region were 2,3‐pentanedione and 1‐phenyl‐1,2‐propanedione, the following compounds being without effect: phenylglyoxal, methylglyoxal, glyoxal, 1,2‐cyclohexanedione and acetone. Of the latter, however, 1,2‐cyclohexanedione, phenylglyoxal, methylglyoxal and acetone inactivated lactoperoxidase at 254 nm.

The butanedione‐sensitized photochemical inactivation of lactoperoxidase was prevented by the presence of free radical scavengers and singlet oxygen quenchers, the best protectors being the histidine derivatives L‐3‐methylthistidine, L‐1‐methylhistidine and L‐thiol‐2‐histidine, which displayed dual quenching ability. No photochemical inactivation off lactoperoxidase occurred in anoxic media even in the presence of high butanedione concentration and strong illumination.

The butanedione‐sensitized photochemical inactivation of lactoperoxidase was accompanied by extensive changes in the ultraviolet absorption spectra of the enzyme. Simultaneously, a dramatic loss in tryptophan fluorescence occurred. All spectral and fluorescence changes were prevented by the above protectors. The sensitized enzyme inactivation was shown to be an all‐or‐none type of reaction; no partially inactivated enzymes existed.

Amino acid analysis of native and butanedione light‐treated lactoperoxidase showed that the treated enzyme contained considerably less of the following amino acids than the native enzyme: methonine, arginine, tyrosine, aspartic acid, serine, glutamic acid. Histidine residues were probably attacked also. Inhibition and modification studies showed that the activity of the enzyme was not based on arginyl and tryptophyl residues.

These results suggest that 2,3‐butanedione, 1,2‐cyclohexanedione, phenylglyoxal and related dicarbonyl compounds, which have previously been used as relatively specific probes for the active arginyl residues of enzymes, cause under suitable modification conditions extensive change in the structure of an enzyme by affecting a multitude of amino acid residues. These compounds can totally inactivate an enzyme regardless of the essentiality or inessentiality of arginyl residues for enzyme activity. The present results warn against drawing conclusions from modification studies with dicarbonyl compounds unless the irradiation conditions are carefully considered.
Preferred Solution Conformation of des‐Arg9‐Bradykinin and Analysis of Structure‐Conformation‐Activity Relationships in the Series [Alan]des‐Arg9‐Bradykinin
Authors: DIVE Vincent , LINTNER Karl , FERMANDJIAN Serge , ST. PIERRE Serge , REGOLI Domenico
Published: March 2005

This paper describes the solution conformation of the vasoactive peptide hormone des‐Arg9‐bradykinin (Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe).

By 1H‐NMR spectroscopy we studied des‐Arg9‐bradykinin and its fragments bradykinin‐(1‐5), bradykinin‐(1‐6) in aqueous solution as a function of pH (titration) and in dimethylsulfoxide solution at two ionic states (cation and neutral ion species).

The preferred solution conformation which is most strongly stabilized in dimethylsulfoxide in the neutral ion species includes a distorted β‐turn II involving the N‐terminal sequence of Pro2‐Pro3‐Gly4‐Phe5 and most likely a C7‐type bend in the C‐terminal part Ser‐Pro‐Phe.

A complete series of analogous ([Alan]des Arg9‐bradykinin, with n= 1,2 … 8) was then investigated by circular dichroism and 1‐H‐NMR spectroscopy in order to study the conformational role played by each residue and to delineate the local and the long‐range effects on conformational brought about by the Xaa→Ala substitutions. Chosen spectral parameters (circular dichroic spectra, chemical shift variations and vicinal coupling constants) characteristic of the preferred solution conformation of the native sequence of des‐Arg9‐bradykinin are followed from analogue to analogue. The important conformational role of the arginine‐1 side chain and its positive charge and the spatial proximity of the N‐terminal and C‐terminal groups, i.e. the folded structure of the peptide can be inferred from these data.

A comparison of the biological activities of the analogues with the conformative perturbations caused by the chemical alterations shows des‐Arg9‐bradykinin conformation and receptor affinity to be equally sensitive to single‐residue substitutions. The correct orientation of the arginine‐1 side chain, the precise geometry of the turn involving residues 2‐5 and of the C‐terminal Pro‐Phe sequence are of primary importance.
Polymerization/Excision Kinetics of Escherichia coli DNA Polymerase I
Authors: SAGHI Mohammed , DORIZZI Mireille
Published: March 2005

The incorporation and excision kinetics of Escherichia coliDNA polymerase I in the replication of (dA)n with dUTP and (dI)n with dCTP was studied at various temperatures and pH. When the incorporation/excision ratio (dy/dx) is plotted against the concentration of deoxynucleoside triphosphate [S] two kinds of curves are obtained. With (dI)n, dy/dx increases with [S], then reaches an asymptotic value. This behaviour, consistent with a kinetic amplification or kinetic proofreading mechanism, is observed at all temperatures and pH. With (dA)n, dy/dx increases with [S] but in a convex, instead of a concave manner. In this case, we approximated the curves by straight lines at the origin, in conformity with the prediction of the frayed‐unfrayed model. Again a single behaviour is consistently observed at all temperatures and pH.

The data were analyzed in terms of ratios or products of three kinetic constants: ki for incorporation, ke for excision and, in the (dI)n system, θ for the processing of the incoming dNTP. The dNMP production in the (dI)n system is dominated by the keθ term which increases with temperature and pH. Temperature and pH. Temperature influences excision more than incorporation. The net result being a linear decrease of ki/ke with temperature. The effect is more pronounced with (dI)n than with (dA)n and is probably related to the stability of the template‐primer complex. The ki/ke term shows a bell‐shaped dependency with pH in the (dI)n system. With (dA)n it remains constant between pH 7.5 and 8.5 then decreases with a transition midpoint at pH 9.0. We suggest that the pH profiles may reflect the ionization of the template in the first case, and of the substrate in the second.
Inhibition of Mitochondrial Protein Synthesis and Energy Coupling by Fragment A of Diphtheria Toxin
Authors: ABRAHAM Abraham K. , FLATMARK Torgeir , TANGERÅS Arild , PIHL Alexander
Published: March 2005

The effect of intact diphtheria toxin and its fragment A on energy‐dependent functions in mouse liver mitochondria/mitoplasts has been studied. Fragment A was found to inhibit protein synthesis in mitoplasts to the same extent (approximately 80%) as the uncoupler carbonylcyanide p‐trifluoromethoxyphenylhydrazone, but had no similar effect in lysed mitoplasts. Intact diphtheria toxin had no effect in either case.

Fragment A was found to function as a potent uncoupler in isolated mitochondria and mitoplasts, inhibiting oxidative phosphorylation by approximately 80% at a concentration (2 μg fragment A/mg of protein) that did not inhibit protein synthesis. In contrast, intact toxin slightly increased the tightness of energy coupling in isolated mitoplasts. 125I‐labelled intact diphtheria toxin was bound to mitoplasts to about the same extent as labelled fragment A.

At concentrations which efficiently inhibited mitochondrial protein synthesis, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides, nor was it capable of ADP‐ribosylating mitochondrial proteins, indicating that the well known enzymatic activity of fragment A is not involved in the observed effect in mitochondria.

The results indicate that fragment A of diphtheria toxin inhibits protein synthesis in mitochondria and mitoplasts by inhibiting mitochondrial energy transduction. The detailed mechanism of the uncoupling effect and its possible significance in intact cells remain to be elucidated.
Cytosolic and Nuclear Binding of 2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin to the AhReceptor in Extra‐Hepatic Tissues of Rats and Mice
Authors: MASON Michelle E. , OKEY Allan B.
Published: March 2005

Induction of aryl hydrocarbon hydroxylase (cytochrome P1‐450) is mediated by the Ah receptor which initially binds inducing chemicals in the cytoplasm, after which the inducer‐receptor complesx translocates into the nucleus. Ah receptor can be detected and quantified by sucrose‐density‐gradient analysis of its binding to 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin. Labeling in vitro of cytosols from C57BL/6J mice and Sprague‐Dawley rats reveals Ah receptor in several tissues. with concentrations in the rank order for C57BL/6J mice: liver > lung ≫ kidney ∼ thymus ∼ small intestine > prostate; for Sprague‐Dawley rats: liver ∼ lung ∼ thymus ≫ small intestine ≫ kidney. Cytosol receptor was not detectable by assays in vitro on skin,skeletal muscle, heart, brain or testis of C57BL/6J mice or Sprague‐Dawley rats, nor was specific receptor detectable in cytosol from any tissue of genetically ‘non‐responsive’ DBA/2J mice. Injection in vitro of 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin led to recovery of specific nuclear Ah receptor from liver, lung and kidney of both genetically ‘responsive’ C57BL/6L mice and ‘non‐responsivr’ DBA/2J mice than in C57BL/6J mice reciving the same dose, and nuclear receptor was detectable only with injection of high doses of 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin. Althouth cytosolic Ah receptor in DBA/2J mice appears to be ‘defective’ and cannot be detected directly by assays in vitro, recovery of specific receptor from nuclei of DBA/2J tissues implies that binding of 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin to Ah receptor does occur in vitro in these animals; this may explain the ability of ‘non‐responsive’ DBA/2J mice to exhibit induction of aryl hydrocarbon hydroxylase when treated with the very potent inducer, 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin.
Positive and Negative cAMP‐Mediated Control of Tyrosine Aminotransferase Synthesis in Reuber H35 Hepatoma Cells
Authors: SNOEK Gerry T. , VOORMA Harry O. , VAN WIJK Roel
Published: March 2005

Induction of L‐tyrosine:2‐oxoglutarate aminotransferase (EC 2.6.1.5) by N6, O2′‐dibutyryl‐adenosine 3′,5′‐ monophosphate (Bt2cAMP) in Reuber H35 hepatoma cells reaches a maximum value between 3‐5 h after addition of Bt2cAMP and subsequently decreases in the continuous presence of Bt2cAMP. We have investigated the kinetics of the increase, i.e. induction, and the decrease, i.e. the repressed state, of the tyrosine‐aminotransferase‐synthesizing system under these conditions.

Our experimental results are as follows. 1. The repressed state of the tyrosine‐aminotransferase‐synthesizing system is not caused by a decrease in the intracellular cAMP concentration. 2. The repressed state is inhibited by actinomycin D (while induction is not inhibited). 3. During the repressed state no effect of dexamethasone on tyrosine aminotransferase synthesis is found, while during induction Bt2cAMP and dexamethasone act synergistically. 4. Longer starvation of the cells in serum‐free medium has no influence on the kinetics of the induction/repressed state curve.

From these results we have concluded that the machanism of the transition to the repressed state of the tyrosine‐aminotransferase‐synthesizing system is essentially different from the mechanism of deinduction which occurs after removal of the inducer. Moreover, the repressed state of the system is a phenomenon which is induced by Bt2cAMP separately from induction at a different level of protein synthesis.
Induction of Ornithine Decarboxylase in Guinea Pig Lymphocytes by the Divalent Cation Ionophore A23187
Authors: OTANI Shuzo , KURAMOTO Ayako , MATSUI Isao , MORISAWA Seiji
Published: March 2005

The divalent cation ionophore, A23187, at a concentration of 0.25 μg/ml, enhanced influx of Ca2+. activity of ornithine decarboxylase and incorporation of [3H]thymidine into DNA of guinea pig lymphocytes. Combined treatment of cells with A23187 and dibutyryladenosine 3′. 5′‐monophosphate (Bt2cAMP) augmented these three events. A23187 at a concentration of 0.06 μg/ml which was insufficient for induction of ornithine decarboxylase stimulated neither Ca2+ influx nor [3H]thymidine incorporation, but stimulated Ca2+ efflux. A23 187 (0.06 μg/ml) in combination with Bt2cAMP caused a marked induction of ornithine decarboxylase and stimulation of [3H]thymidine incorporation into DNA. When the time of Bt2cAMP addition was delayed after A23187, the stimulation of ornithine decarboxylase activity decreased. Washout of Bt2cAMP from cell culture earlier than 4 h of incubation caused a reduction in the stimulatory effect of Bt2cAMP. These results suggest that raising concentrations of cytoplasmic Ca2+ and cellular cAMP are important to some initial events leading to induction of ornithine decarboxylase and these biochemical changes are obligatory sequential steps for stimulation of DNA synthesis.
Analysis of Mycolic Acids from a Group of Corynebacteria by Capillary Gas Chromatography and Mass Spectrometry
Authors: GAILLY Chantal , SANDRA Patrick , VERZELE Maurice , COCITO Carlo
Published: March 2005

The cell wall of leprosy‐derived corynebacteria (a group of ‘diphtheroids’ isolated from human leprosy lesions and patients' blood) was previously shown to contain, in addition to peptidoglycan and arabinogalactan, mycolic acids. These α‐branched β‐hydroxy fatty acids were attributed to the corynomycolic group, according to their RF in monodimensional thin‐layer chromatography. In the present work, mycolic acids from leprosy‐derived and reference corynebacteria have been fractionated by monodimensional and bidimensional thin‐layer chro‐ matography and by gas chromatography. Pyrolyzed mycolic acids have been analysed on conventional packed columns, whereas intact methyl esters of mycolic acids with free and silylated β‐hydroxyl group have been analyzed on capillary columns, and their structure has been established by mass spectrometry.

In all leprosy‐derived corynebacteria, some 20 components containing 24‐ 36 carbon atoms and 0‐4 double bonds were obtained. The three major groups had 32, 34 and 36 carbons, and the frequency of unsaturated versus saturated chains increased proportionally to the molecular weight. For comparison, the main components of a reference corynebacterium, Corynebacterium diphtheriae PW8, had 30 and 32 carbons, and their hydro‐ carbon chains were essentially saturated. This work confirms the relative chemical homogeneity of different leprosy‐derived corynebacteria and describes some peculiar traits in the chemical structure of this group of organisms. In addition, it shows the complexity of the mycolic acid fraction of corynebacterial cell wall and suggests that the mycolic acid pattern is a sort of fingerprint of each bacterial strain grown under standard conditions. Finally, the fractionation of intact corynomycolic acid methyl esters with free or silylated β‐hydroxyl group by capillary gas chromatography proved to be the best analytical procedure at present available for resolving this complex mixture of corynomycolate isomers. Structural determination of silylated samples by mass spectrometry is preferred because they have more diagnostic fragments.
Agonist‐Mediated Conformational Changes of Muscarinic Receptors in Rat Brain
Authors: VAUQUELIN Georges , ANDRÉ Claudine , DE BACKER Jean‐Paul , LADURON Pierre , STROSBERG A. Donny
Published: March 2005

Muscarinic acetylcholine receptors were identified in the microsomal P fraction of rat forebrain by the specific binding of the radiolabeled antagonist [3H]dexetimide. Binding occurred to a single class of non cooperative sites (3.25 μmol/mg protein) with an equilibrium dissociation constant of 1.1 nM. Agonist/[3H]‐ dexetimide competition binding experiments allowed the distinction between two major muscarinic receptor subpopulations, having respectively high affinity (20 % of the total receptor population) and low affinity for agonists, but with the same affinity for antagonists. A 610‐fold difference in affinity was calculated for carbamoyl‐ choline, the agonist extensively investigated in this study.

The alkylating reagent N‐ethylmaleimide did not affect the total receptor number, antagonist binding to the high‐affinity and low‐affinity sites, nor agonist binding to the high‐affinity sites. The reagent, however, caused a net increase in agonist affinity for the low‐affinity sites. This process was dependent on time and dose of N‐ethylmaleimide, until a maximal increase in affinity (fourfold increase for carbamoylcholine) was attained. This suggests a quanta1 conversion of the low‐affinity sites by the reagent into an alkylated form, which possesses a higher affinity for agonists but an unchanged affinity for antagonists. The rate of alkylation was enhanced by the presence of agonists but not of antagonists, which is indicative for the ability of agonists to mediate a conformational change of these sites. The close correlation between the N‐ethylmaleimide‐mediated increase in drug affinity for the low‐affinity sites and the ability of the drugs to enhance alkylation of these sites by N‐ethylmaleimide can be explained by the ability of (a) muscarinic drugs to interact with the low‐ affinity sites according to the Monod‐Wyman‐Changeux ‘Plausible Model’ and (b) N‐ethylmaleimide to freeze these sites in the ‘active’ conformation by alkylation.
On the Properties of Immobilized Elongation Factor Tu from Thermus thermophilus HB8
Authors: FISCHER Wolfgang , DERWENSKUS Karl‐Heinz , SPRINZL Mathias
Published: March 2005

Elongation factor Tu from Thermus thermophilus was coupled to cyanogen‐bromide‐activated Sepharose 4B and its properties were investigated. The immobilized elongation factor retained its ligand binding properties. It specifically binds GDP and GTP but does not interact with other nucleotide 5′‐phosphates. A conversion of immobilized EF‐Tu. GDP to EF‐Tu. GTP can be achieved by simple equilibration with GTP. The immobilized EF‐Tu. GTP specifically binds aminoacyl‐tRNAs and allows a facile purification of aminoacyl‐tRNA species from bulk tRNA. It is stable at room temperature for several months and can be repeatedly used for amino‐ acyl‐tRNA isolation.
Purification and Characterization of the CytochromecOxidase fromRhodopseudomonas sphaeroides
Authors: GENNIS Robert B. , CASEY Robert P. , AZZI Angelo , LUDWIG Bernd
Published: March 2005

When grown aerobically in the dark,Rhodopseudomonas sphaeroidesdevelops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed. The aa3‐type cytochrome c oxidase fromRps. sphaeroideshas been purified in Triton X‐100 by affinity chromatography with Sepharose 4B coupled to yeast cytochromec. The oxidase contains 14 nmol heme a/mg protein and is composed of three polypeptide subunits with relative molecular masses of 45000, 37000 and 35000. The enzyme is highly active in the presence of detergents, with a maximal velocity of 300s−1/mol oxidase using either yeast or horse‐heart cytochromec.

TheRps. sphaeroides oxidaseis cross‐reactive with antibodies directed against the oxidases fromParacoccus denitrificansandSaccharomyces cerevisiae. A particularly close relationship is indicated in the case ofP. denitrificans. TheRps. sphaeroidesoxidase has been incorporated into phospholipid vesicles. The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5. Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed. The data suggest that theRps. sphaeroides oxidasedoes not function as a proton pump.
The Reaction of Bovine Glutamate Dehydrogenase with Periodate‐Oxidised ADP
Authors: FAVILLA Roberto , BAYLEY Peter M.
Published: March 2005

The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding.

The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd∼ 12 μM (20°C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm.

This complex is unstable and decays with a half‐life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme.

Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit.

Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or l‐hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NADNAD+ alone.

Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity.

The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site.

Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site.
Somatic Antigens of Pseudomonas aeruginosa
Authors: DMITRIEV Boris A. , KOCHAROVA Nyna A. , KNIREL Yuriy A. , SHASHKOV Alexander S. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M.
Published: March 2005

An O‐antigenic lipopolysaccharide, manifesting a high serological activity in the reaction of passive haemag‐glutination and its inhibition, was isolated from Pseudomonas aeruginosa O:6 cells (Lanyi). Splitting‐off of the lipid component by mild acid hydrolysis resulted in the polysaccharide deprived of O‐specific activity.

The polysaccharide chain of the lipopolysaccharide was made up exclusively of the residues of amino sugars, among which have been identified: 2‐acetamido‐2‐deoxy‐d‐galactose (GalNAc), 2‐acetamido‐2,6‐dideoxy‐d‐ glucose (QuiNAc), 2‐acetamido‐2,6‐dideoxy‐d‐galactose (FucNAc) as well as 2,3‐diacetamido‐2,3‐dideoxy‐d‐glucuronic acid [Glc(NAc)2A] found naturally for the first time. The principal methods of establishing the polysaccharide structure were 13C nuclear magnetic resonance, methylation analysis and solvolysis with hydrogen fluoride. Depending on solvolysis conditions, a disaccharide or a trisaccharide containing the diacetamidouronic acid residue was formed.

From the results obtained it followed that the polysaccharide chain of the O‐antigenic Ps. aeruginosa O:6 lipopolysaccharide represents an acidic hexosaminoglycan constructed of the repeating tetrasaccharide units of the following structure:→4)dGalNAc(αl→4)dGlc(NAc)2A(β1 →3)dFucNAc(α1→3)dQuiNAc(α1→.
The Surface‐Exposed, Trypsin‐Accessible Segments of Apolipoprotein B in the Low‐Density Lipoprotein of Human Serum
Authors: CHAPMAN M. John , MILLET Anne , LAGRANGE Dominique , GOLDSTEIN Sonia , BLOUQUIT Yves , TAYLAUR C. Emlyn , MILLS Gervase L.
Published: March 2005

Limited tryptic digestion of native low‐density lipoprotein (LDL) (ϱ 1.024‐ 1.045 g/ml) liberates some 20 ‐ 25% of its surface‐exposed protein as soluble, lipid‐free peptides (T‐peptides). The T‐peptides were resolved into twelve subfractions (peaks 1 ‐ 12) by gel filtration chromatography on Bio‐Gel P4 in 5 % formic acid, and classified into four major groups on the basis of criteria of size, amino acid composition and predominant C‐terminal residue. The first group, peaks 1‐4, (∼ 30% of total peptide protein; M, 2600‐6000), displayed lysine as preponderant C‐terminal (Lys : Arg, 3.7‐7.3:1), were acidic in nature (basic:acidic residues, 0.37‐0.61:1), and more hydrophobic than holo‐apo‐LDL. Peaks 1 and 2 contained glycopeptides (four or less by fingerprinting), the monosaccharide composition of which resembled that of LDL glycopeptides of type I1 [Swaminathan, N. & Aladjem, F. (1976) Biochemistry, 15, 1516‐15211. The second group, peaks 5 and 6, (∼ 30% of the total; Mr, 1500‐2000), were also apparently derived predominantly by cleavage at lysyl peptidyl bonds (Lys:Arg, 4.6‐4.9:1), but were distinct in their higher proportion of basic residues (acidic:basic, 0.83‐1.05:1). The third group (peaks 7 and 8) contained peptides approx. eight residues in length, with equal proportions (∼ 10%) of arginine and lysine. In contrast, the smaller fragments (2‐5 residues) of peaks 9‐12 exhibited arginine as major C‐terminal residue (Lys:Arg, mean 0.45:1), as well as similar amounts of acidic and basic residues. Examination of the quantitative distribution of lysine and arginine showed 80% of arginines to be present in fragments of eight residues or less, with the majority (45 %) in peptides of 2‐5 residues. In contrast, ∼ 70% of total lysines were located in fragments of z 10‐20 residues; only ∼ 10% of lysines were in small (2‐5 residue) peptides. Arginine appears therefore to occur most often at short (2‐5 residues) and lysine at longer (six or more residues) distances from the next lysine or arginine in trypsin‐accessible LDL protein. Clusters of a minimum of two positive charges, often featuring arginine, can thus be envisaged. The resemblance of the amino acid profiles of the major peptide subfractions (peaks 1‐8) to that of apolipoprotein B further supports the contention that this apoprotein possesses a repetitive‐type sequence. Finally, despite evaluation of peptide heterogeneity by fingerprinting, the inferred presence of incompletely‐cleaved tryptic peptides in peaks 3, 4 and 5 precludes attribution of a minimal unique amino acid sequence to apolipoprotein B.
A Comprehensive Approach to the Study of Collagen Primary Structure Based on High‐Performance Liquid Chromatography
Authors: VAN DER REST Michel , FIETZEK Peter P.
Published: March 2005

A comprehensive approach for the structural microanalysis of collagen based on high‐performance liquid chromatography (h.p.1.c.) has been developed using calf skin type I collagen as a model system. The α, β and γ components were separated, after heat denaturation, on a TSK 4000 SW gel permeation column, using a nonvolatile buffer. Monitoring at 210 nm permits the detection of 1 μg of a single chain. The α1(I) and α2(I) chains were completely resolved using a large‐pore reversed‐phase column (Vydac 201 TP 4.6) eluted by an aqueous acetonitrile gradient (24‐48 %) containing 0.01 M heptafluorabutyric acid as an ion‐pairing agent. The purified α1(I) chain was digested with CNBr and the resulting fragments separated in the same chromatography system with a gradient containing a 12.8‐44.8%, acetonitrile gradient. The purified α1(I)CB 3 peptide was further cleaved with trypsin and the resulting peptides separated first by a similar chromatography with a 4‐32 % acetonitrile gradient. Resolution of some poorly separated peptides was obtained by a rechromatography using trifluoroacetic acid as counterion. The isolated peptides were hydrolyzed and identified by their amino‐acid composition. Sequencing of h.p.l.c.‐purified α1(I)CB 3 was also performed to demonstrate the suitability of the technique for the preparation of peptides for amino‐acid sequencing. This study demonstrates that detailed structural analysis can be performed on 3 mg of a purified collagen.
The Major Cell Envelope Protein of Micrococcus radiodurans (R1)
Authors: BAUMEISTER Wolfgang , KARRENBERG Franck , RACHEL Reinhard , ENGEL Andreas , TEN HEGGELER Beatrice , SAXTON W. Owen
Published: March 2005

The so‐called HPI layer, representing the major cell envelope protein of the radiotolerant bacterium Micrococcus radiodurans, is a hexagonally patterned macromolecular monolayer; it is intimately associated with the outer membraneous layer (‘backing layer’) of the bacterium and covers its surface completely. By virtue of its quite unusual resistance to chemical perturbants it can easily be isolated, e.g. by detergent extraction of whole cells.

A detailed description of the morphology of the HPI layer protein is given (resolution 1.8 nm), based on electron microscopy and image processing. Infrared spectroscopy shows that the secondary structure of the protein is stable under a wide range of environmental conditions; a considerable portion (30%) of the protein exists in the form of B structure.

Direct mass measurements in a scanning transmission electron microscope reveal that the HPI layer protein has a molecular mass of approximately 655 kDa. Although masked by extensive proteolytic cleavage it appears that the morphologicaI protein units are composed of six identical protomers. All evidence accumulated so far supports our view that proteolysis is not a preparation artifact, but occurs already in vivo, without affecting the physical structure of the protein.
The Primary Structure of Escherichia coli K12 2‐Deoxyribose 5‐Phosphate Aldolase
Authors: VALENTIN‐HANSEN Poul , BOËTIUS Finn , HAMMER‐JESPERSEN Karin , SVENDSEN Ib
Published: March 2005

The sequence of the deoC gene of Escherichia coli K12 and the amino acid sequence of the corresponding protein, deoxyriboaldolase, has been established. The protein consists of 259 amino acids with a molecular weight of 27 737. The purified enzyme may exist both as a monomer and as a dimer. On the basis of amino acid composition, molecular weight and catalytic properties, the enzymes from E. coli and Solmonella typhimurium seem to be almost similar. They belong to the class I aldolases, which form Schiff base intermediates. Using data for the S. typhimurium enzyme, the lysine residue involved in the active site in the E. coli enzyme was tentatively identified.