bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ribonuclease Activity in Escherichia coli Polyribosomes
Authors: Mangiarotti Giorgio , Turco Emilia 
Published: March 2005

Polyribosomes containing mRNA differentially labelled with [3H]uracil and [14C] uracil in the 5′‐ and the 3′‐proximal protions were isolated from Escherichia coli cells. mRNA degradation to alcohol‐soluble products occurred when polyribosomes were incubated in conditions of protein synthesis and proceeded in the 5′ to 3′‐direction. 15% of the mRNA was degraded at the end of a 10‐min period of incubation.The mRNA‐degrading enzyme (or enzymic system) was found to be bound to polyribosmes; it could be removed by washing in low salt. The content in RNAase activity per ribosomes in polyribosomes increased three to five‐fold when polyribosomes were isolated from cells in which extensive run‐off of ribosomes from mRNA had been induced. On the contrary the RNAase activity in free ribosomes was not affected. This is taken as an indication that the enzyme(s) responsible for the degradation of endogenous mRNA in vitro is not aspecifically adsorbed to ribosomes, but specifically located towards the 5′‐end of the mRNA chains in polyribosomes.
The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides
Authors: Berezin Ilya V. , Kazanskaya Novella F. , Klyosov Anatole A. , švedas Vytautas K. 
Published: March 2005

The applicability of the acylenzyme mechanism to the tryptic hydrolysis of the amide bond has been studied. It was found that trypsin acetylated at the hydroxyl group of the “active” serine or acetyl (Ser‐183)trypsin displays no activity with respect to both esters (N‐benzoyl‐l‐arginine ethyl ester and p‐nitrophenyl‐p′‐guanidinobenzoate) and anilides (N‐carbobenzoxy‐l‐arginine m‐nitroanilide and N‐benzoyl‐dl‐arginine p‐nitroanilide). It was also found, that deacetylation of acetyl(Ser‐183)trypsin is effectively promoted by N‐carbobenzoxy‐dl‐arginine m‐nitroanilide (Z‐Arg‐Nan) taken at a high concentration. As was found by means of a detailed kinetic analysis of experimental results, the binding of this anilide with acetyl(Ser‐183)trypsin increases the rate constant of deacylation of the latter by as many as 23 times. This effect which results in a rapid increase in the concentration of native trypsin in the reaction system, causes a marked increase in both kcat and Km(app) of tryptic hydrolysis of Z‐Arg‐Nan). Apparently, the phenomena is the main reason for the erroneous conclusions of Bresler et al. (1969, 1971), who stated: trypsin with an acylated active serine remains a highly efficient catalyst of amide hydrolysis. Finally, the data of Bresler et al. on tryptic hydrolysis and hydroxylaminolysis which contradict the acylenzyme mechanism of hydrolysis of the amide bond can be readily accounted for by chemistry of hydroxylamine, as was done by Bender et al. (1964) and Epand and Wilson (1963).Thus, the data obtained so far strongly support the acyl‐enzyme mechanism of the hydrolysis of the amide bond catalyzed by trypsin and α‐chymotrypsin.
Specific Binding of Phosphate by a Chloroform‐Soluble Protein from Rat‐Liver Mitochondria
Authors: Kadenbach Bernhard , Hadváry Paul 
Published: March 2005

If an aqueous solution of [32P]phosphate is shaken with a chloroform‐extract of rat liver mitochondria, the label is bound specifically by a number of chloroform‐soluble proteins.
Several chloroform‐soluble proteins contain free SH‐groups, as indicated by the binding of [14C]ethacrynate, a specific reagent for SH‐groups.
One fraction of chloroform‐soluble proteins binds phosphate in high amounts, and this binding of phosphate is specifically inhibited by SH‐reagents.
Among various labeled anions (phosphate, malate, citrate, glutamate and ADP) only phosphate was shown to be bound at a significant amount by a chloroform‐extract of rat liver mitochondria.
A Highly Sensitive Method for Amino‐Acid Analysis by a Double‐Isotope‐Labelling Technique Using Dansyl Chloride
Authors: Brown Joseph P. , Perham Richard N. 
Published: March 2005

A method for amino acid analysis by double isotope labelling has been developed using 3H‐labelled dansyl chloride and 14C‐labelled amino acids as internal standards.
As little as 20 pmol of an amino acid can be accurately measured by this method.
The amino acid compositions of several peptides, determined using about 50 pmol peptide, are reported and compared with the results obtained using conventional high‐sensitivity ion‐exchange chromatography. The correspondence is very good.
The amino acid composition of a tryptic peptide containing the active site disulphide bridge of the lipoamide dehydrogenase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been shown to be identical to that of the corresponding peptide from the 2‐oxoglutarate dehydrogenase complex of the same organism. This result is consistent with the view that the lipoamide dehydrogenase components of both complexes have a common amino acid sequence.
Changes of the Fluorescence of l‐Anilino‐8‐naphtalenesulfonate, Associated with the Membranes of the Sarcoplasmic Reticulum, Induced by General Anesthetics
Authors: Augustin Jan , Hasselbach Wilhelm 
Published: March 2005

The fluorescence of 1‐anilino‐8‐naphthalene sulfonate (ANS), dissolved in buffer, is enhanced by the narcotics ether and halothane.Ether and halothane considerably reduce the quantum yield of ANS, bound to native and lipid‐depleted sarcoplasmic vesicles. Obviously the effect of the anesthetics takes place at the same sites before and after delipidation.The excitation spectrum of ANS, associated with the sarcoplasmic membranes, is resolved by halothane into at least two maxima with a new separate maximum at 394 nm. The emission spectrum is not influenced.The narcotics are only able to influence the fluorescence of those ANS molecules which can be displaced by oleic acid, a noncompetitive inhibitor of the interaction of the dye with the membranes of native and lipid‐depleted vesicles.Higher homologues of diethylether did not influence the fluorescence. Fluorescence quenching by ether and halothane is enhanced with rising temperatures. The polarisation of the fluorescence of membrane‐bound ANS is enhanced by halothane.The intrinsic fluorescence of native and lipid‐depleted vesicles is considerably decreased by halothane, hardly influenced by ether. Concentrations of 2.2 M sucrose completely abolish the fluorescence quenching by the narcotics.All effects produced by ether and halothane are reversible.
The Dynamics of Nucleic‐Acid Single‐Strand Conformation Changes
Authors: Pörschke Dietmar 
Published: March 2005

The dynamics of the single‐strand conformation changes of oligo‐ and polyriboadenylic acids has been investigated by a fast temperature‐jump technique. Chemical rexalation processes have been observed in the range of 0.1 to 1 μs. The relaxation times are independent of the nucleotide concentration and also do not depend upon the pH within the range 6.9 to 9.2. The amplitudes correspond to those expected from thermal melting curves. According to these results the relaxation is assigned to the “single‐strand helix‐to‐coil” transition.
Measurements af different wavelengths show that the relaxation times observed in the spectral range 240 nm to 270 nm are different from those observed around 285 nm by a factor of 2 to 3. This result demonstrates that the “helix‐coil” transition of adenylic acid chains cannot be described by a simple all‐or‐none model with two reaction states.
As expected for a single‐strand helix‐coil transition there is a relatively small ionic strength dependence: a decrease of the salt concentration leads to an increase of the relaxation time. At high ionic strengths of monovalent ions (1 M and more) the appearance of relaxation times at 5 to 10 μs and in the ms range seem to indicate aggregation.
The presence of Mg2+ leads to a strong decrease of the amplitude around 285 nm, whereas the amplitude around 245 nm remains almost unchanged; the time constant of the 245‐nm relaxation increases with increasing Mg2+ concentration. Apparently Mg2+ ions induce a specific shift of the riboadenylic acid chain conformation.
Measurements of the temperature dependence in the range of 0 to 50°C revealed an increasing rate of the conformation change with increasing temperature. The relaxation process measured around 245 nm is characterised by a higher apparent activation enthalpy than that measured around 285 nm.
The relaxation behaviour found for the polymer is also shown by oligoriboadenylic acids. The chain length dependence of the relaxation times is relatively small. The difference of the relaxation behaviour observed around 245 nm and around 285 nm extends to the dimer. Thus the observation of two different relaxation times cannot be explained by a cooperative effect.

The relaxation time constants observed for oligo‐ and polyriboadenylic acids indicate the existence of energy barriers in the conformational transition. Similar time constants have been observed previously for the elementary process of base pairing in adenylic‐uridylic acid double helices.The observation of two relaxation time constants for the conformation change of riboadenylic acid chains demonstrates the existence of more than two different conformation states.
The Primary Structure of the Porcine Luteinizing‐Hormone α‐Subunit
Authors: Maghuin‐Rogister Guy , Combarnous Yves , Hennen Georges 
Published: March 2005

The primary structure of porcine luteinizing hormone α‐subunit was established by determination of the amino acid sequence of its tryptic peptides, which were aligned by homology with the known sequence of ovine luteinizing hormone α‐subunit. Its polypeptide chain is shorter by six amino acid residues at its amino‐terminus, when compared to the ovine α‐subunit. Furthermore four amino acid replacements are observed in the porcine as compared with the ovine chain.Heterogeneity of both carbohydrate prosthetic groups of porcine luteinizing hormone α‐subunit is indicated by the different sugar compositions of the glycopeptides isolated in the course of this work.
Further Evidence for the Pool Function of Ubiquinone as Derived from the Inhibition of the Electron Transport by Antimycin
Authors: Kröger Achim , Klingenberg Martin , Schweidler Sabine 
Published: March 2005

The role of ubiquinone (Q) in the respiratory chain is studied by analyzing the inhibition by antimycin of the electron transport in uncoupled submitochondrial particles. As a result, the antimycin inhibition curve of substrate oxidation is quantitatively explained.

The participation of Q in the electron transport is required for the occurrence of nonlinear antimycin inhibition curves as demonstrated with external hydroquinones of different redox potentials as hydrogen donors. The respiration is non‐linearly inhibited with a hydroquinone of sufficiently low redox potential to reduce the mitochondrial Q (menadiol) whereas a linear inhibition curve is found with hydroquinones of higher redox potentials (durohydroquinone, dimethoxy‐dimethylbenzohydroquinone). If Q is extracted, the inhibition curve also with menadiol becomes linear. This demonstrates the relation of the nonlinearity of the inhibition curve to the function of Q as a distributor of hydrogen between parallel cytochrome chains.
The electron transport through the cytochrome segment is inhibited linearly with increasing amounts of antimycin as demonstrated by the linear titration of three different functions: (a) the oxidation rate of Qred on addition of oxygen to anaerobic submitochondrial particles, (b) the repiratory activity with added hydroquinones, (c) the acceptor activity as defined by the ratio of the respiration rate to the redox state of Q (Kröger, Klingenberg and Schweidler, 1973).
The ratio of donor to acceptor activity to Q (Vred/Vox) is the main parameter in controlling the shape of the antimycin titration curve. This curve becomes more nonlinear with decreasing this ratio. Also the redox state of Q is nonlinearly dependent on the amount of antimycin and controlled by the donor/acceptor activity ratio.
A theoretical hyperbolic relation of the rate of substrate oxidation to the amount of antimycin is derived, involving the ratio donor to acceptor activity and the linear inhibition by antimycin of the electron transport through the cytochrome segment. With independently measured donor/acceptor activity ratios, antimycin titration curves are calculated which give a good fit to the experimental titrations over the range from highly nonlinear to almost linear curves. The observed nonlinearity is thus recognized to result from a hyperbolic relationship. Also the calculated titration curves for the redox state of Q are in agreement with the experiments.
The deviation of the experiment from the calculated curves near equimolarity between the amount of antimycin and inhibition sites can be accounted for by the dissociation of antimycin from the site of inhibition.
Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling
Authors: Roustan Claude , Brevet Annie , Pradel Louise‐Anne 
Published: March 2005

The environment of the active site of creatine kinase has been investigated by means of three chromophoric reagents: 2‐hydroxy‐5‐nitrobenzyl bromide, 2‐methoxy‐5‐nitrobenzyl bromide and 2‐(dansylamino)ethyl monophosphate.

The first two reagents are incorporated at the essential thiol groups. The effect of pH on the spectra of the 2‐hydroxy‐5‐nitrobenzyl‐labelled creatine kinase suggests that this reagent lies inside the active site near an ionized residue, while the 2‐methoxy‐5‐nitrobenzyl group is apparently exposed to the medium. Conformational changes induced by the binding of different ligands, including nucleotide substrates or anions, produce only perturbations of the 2‐hydroxy‐5‐nitro‐benzyl chromophore.
The 2‐(dansylamino)ethyl monophosphate (which is really an analogue of the nucleotide substrate) interacts with the nucleotide site of creatine and arginine kinases in a relatively hydrophobic environment.
β‐Chain Allostery in the Frozen Quaternary T‐Structure of Haemoglobin M Iwate
Authors: Gersonde Klaus , Overkamp Matthias , Sick Hinrich , Trittelvitz Eberhard , Junge Wolfgang 
Published: March 2005

Two hybrid species of haemoglobin M Iwate exist: α2Mmetβmetβdeoxy and α2Mmetβ2deoxy These species differ in their ligand and effector binding properties.The α2Mmetβmetβdeoxy hybrid is characterized by a Bohr effect, while the Hill coefficient is n= 1.00. The energy of the interaction between the Bohr protons and the ligand‐binding site corresponds to the interaction energy found in monomeric haemoglobins and amounts to 0.55 kcal/mol. One molecule of 2, 3‐bisphosphoglycerate per mole is bound at neutral pH, while at alkaline pH two co‐operative molecules are bound. Binding of 2,3‐bisphosphogly‐cerate reduces the ligand affinity and shifts the pK value of the Bohr proton binding site to the alkaline region.The α2Mmetβ2deoxy hybrid shows a pH‐dependent co‐operativity of the ligand‐binding sites (n= 1.8 at pH 9). The co‐operativity is weakened by binding of protons or 2,3‐bisphospho‐glycerate. The latter lowers the ligand affinity, but in the alkaline region no influence is observed. Furthermore the Bohr effect curve is shifted to the alkaline region by this compound but the amplitude remains unchanged.The results of the present paper are interpreted in terms of intra‐chain and β‐β inter‐chain interactions, the latter being responsible for a consecutive change of the ligand affinity in a “frozen” T‐state molecule.
On the Reactivity of the Thiol Group of Thiolsubtilisin
Authors: Polgár László , Halász Piroska , Moravcsik Ernó 
Published: March 2005

The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH‐range the –SH group of the enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in 2H2O it is concluded that a mercaptide‐imidazolium ion‐pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH.
Iodoacetamide reacts with the –SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo‐, chloro‐, and fluoroacetamide. This might be due to the less polar environment around the –SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo‐ and chloroacetamide in the presence of dioxane.
By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH · 9, whereas the overall protein structure is stable up to pH · 11. D‐2‐Bromo‐n‐butyramide reacts with thiolsubtilisin about 60 times as fast as D‐2‐bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala‐152 and Thr‐220 of thiolsubtilisin may form van der Waals interactions with D‐2‐bromo‐n‐butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D‐2‐bromopropionamide and thiolsubtilisin.
Studies on the Metabolism of Steroids in the Foetus
Authors: Plasse Jean‐Claude , Lisboa Belisário Penna 
Published: March 2005

The metabolism of testosterone in human foetal adrenal glands at mid‐gestation was investigated in vitro. After incubations with microsome preparations in a medium supplemented with NADPH‐regenerating system, the metabolites were isolated by thin‐layer and gas‐liquid chromatography, and identified by gas chromatography‐mass spectrometry. 4‐Androstenedione, 3β‐hydroxy‐4‐androsten‐17‐one and 4‐androstene‐3α (and 3β),17β‐diols were identified in both incubation series.The presence of 1β‐, 2α‐, 2β‐, 6α‐ and 6β‐hydroxylase activities in foetal adrenal glands was demonstrated by the isolation of the corresponding testosterone derivatives. The biosynthesis of these steroids in human adrenal gland has not been demonstrated before.Because 3β‐hydroxy‐4‐androsten‐17‐one and epimeric 4‐androstene‐3α (and 3β), 17β‐diols could be isolated in these incubation studies the presence of 3α, 3β‐ and 17β‐hydroxysteroid oxidoreductase in the adrenal gland preparation is very likely. Neither 5α(H)‐ nor 5β(H)‐ring reductase activities have been detected.
Studies on the Conformational Changes of Plasminogen Induced during Activation to Plasmin and by 6‐Aminohexanoic Acid
Authors: Sjöholm Ingvar , Wiman Björn , Walléan Per 
Published: March 2005

The hydrodynamic properties of plasminogen are highly affected during the activation process as found by studies on the sedimentation‐velocity, sedimentation‐equilibrium behaviour, gelfiltration behaviour of plasminogen A, plasminogen B and of plasmin inactivated by diisopropylphosphorofluoridate (Dip‐plasmin). The molecular weight was estimated to 92 000, 84000 and 81 000 for plasminogen A, B and Dip‐plasmin, respectively and the frictional ratio, f/f0, was found to be 1.50, 1.56 and 1.55.The circular dichroism (CD) spectra of human plasminogen A, plasminogen B and Dip‐plasmin have been studied at pH 7.4 in 0.1 M KCI. The far‐ultraviolet spectra are almost identical for all the samples and indicate that they contain a large proportion of random structure (about 80%) and about 20%β‐structure, while no significant amounts of α‐helix could be detected. In the near‐ultraviolet region, large significant differences were seen, which are consistent with a stepwise activation of plasminogen A via plasminogen B to plasmin.The effects of 6‐aminohexanoic acid and l‐lysine on the CD spectra of plasminogen A and of the activation peptide on that of plasminogen B indicate that the amino‐terminal peptidic material has great affinity for the rest of the protein at pH 7.4 and greatly affects the CD spectrum between 250–280 nm. In this area, the spectra of Dip‐plasmin and plasminogen B are almost identical and the same spectrum is also obtained with plasminogen A when treated 0.01 M 6‐aminohexanoic acid or 0.1 M l‐lysine. The conformation around the amino‐terminal part of Dip‐plasmin and around the internal peptide bond cleaved during the activation to plasmin seems to be of great importance for the CD spectra at 280–305 nm.
Studies on the Properties of Fish Hemoglobins
Authors: Giardina Bruno , Brunori Maurizio , Binotti Ines , Giovenco Silvia , Antonini Eraldo 
Published: March 2005

The ligand‐binding kinetics of the isolated component from trout (Salmo irideus) blood has been extensively investigated; it appears to be fully consistent with their equilibrium behaviour, described previously. At alkaline pH (∼ 7.5), in a region where the Root effect is not operative, comparison of the kinetics of the various components (Hb trout I, II and IV) shows considerable similarities between them. For Hb trout I (and II) the time course of the reaction with both O2 and CO is practically pH independent. On the other hand Hb trout IV, characterized by a Root effect, displays a strong dependence on pH of the kinetics of ligand binding. Thus in the pH region where the Root effect becomes operative there is a large change of the character, as well as of the speed, of the kinetics with both O2 and CO.The Root effect characteristic of Hb trout IV appears to originate, from a kinetic point of view, in a pH‐dependent change of both the “on” and “off” constants, although at least for O2 the effect of pH on the “off” kinetic constants is much more marked.
5‐Aminolevulinic‐Acid Synthetase of Rhodopseudomonas spheroides Y
Authors: Fanica‐Gaignier Michèle , Clement‐Metral Jenny 
Published: March 2005

The results of initial velocity studies on the 5‐aminolevulinic acid synthetase reaction and product inhibition studies are consistent with an ordered bi‐bi mechanism in which glycine binds to the enzyme first and 5‐aminolevulinic acid dissociates last. The results are confirmed by the spectral changes of holoenzyme caused by the substrates.5‐Aminolevulinic acid synthetase activity is inhibited only by adenosine triphosphate, guanosine triphosphate and pyrophosphate. This inhibition is competitive with glycine and non‐competitive with succinyl‐CoA. Experiments with [α‐32P]ATP, [γ‐32P]ATP and [32P2]pyrophosphate have shown that adenosine triphosphate and pyrophosphate are bound on each of both forms of 5‐aminolevulinic acid synthetase. The ratio is one mole per mole of enzyme. The estimation of –SH groups with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) after binding of inhibitor shows that one –SH is masked. After p‐hydroxymercuribenzoate action on the enzyme, 5‐aminolevulinic acid synthetase does not bind anymore [32P]ATP and is completely inactive. We suggest that ATP and pyrophosphate inhibit 5‐aminolevulinic acid synthetase by acting on an –SH group of the active center of 5‐aminolevulinic acid synthetase.