bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Actinomycin D, Toyocamycin and Cycloheximide on the Synthesis of Low‐Molecular‐Weight Nuclear RNAs in HeLa Cells
Authors: Hamelin Richard , Larsen Christian J. , Tavitian Armand 
Published: March 2005

The effects of three antibiotics on the synthesis of the small‐molecular‐weight nuclear RNA (called small nRNA) have been investigated. These antibiotics have been chosen for their different mechanism of action on RNA synthesis. From these investigations the following observations can be made.a) Low concentrations of actinomycin D, whose effect on the synthesis of nuclear‐precursor ribosomal RNA is well known, abolish the synthesis of the 7‐S rRNA or E peak (or 28‐S‐associated rRNA). Amounts ten times higher are not sufficient to inhibit completely the synthesis of other small nRNAs although the A, C and D peaks and the methylated species of the G and G' peaks are much more inhibited than the 5‐S rRNA.b) Toyocamycin also depresses the synthesis of the 7‐S rRNA or E peak. It does not inhibit however the synthesis of the other nucleoplasmic peaks such as the C, D and G‐G' species. It seems to exert an indirect effect on the nucleolar A, B and H species of small nRNAs at the same time as it blocks the conversion of the 45‐S precursor rRNA into the mature 28‐S and 18‐S rRNA.c) The mechanism of action of cycloheximide on small nRNAs synthesis was investigated both in monolayers and suspension cultures. Although protein synthesis was completely inhibited in both cases, effects on small nRNAs were different: a much greater degradation of various classes of RNA was observed when suspension cultures were used. It is suggested that this degradation was due to an instability of some species of newly synthesized RNA upon cycloheximide treatment.
Metal‐Combining Properties of Human Lactoferrin
Authors: Teuwissen Bruno , Masson Pierre L. , Osinski Pierre , Heremans Joseph F. 
Published: March 2005

When iron · lactoferrin and apolactoferrin were submitted to nitration with tetranitromethane, the number of unmodified tyrosine residues recovered upon acid hydrolysis was 4–5 in the former case against 0–1 in the latter. The iron‐binding capacity of lactoferrin appeared to remain intact following nitration of the iron‐containing form of the protein, whereas metal‐combining properties were destroyed when apolactoferrin was treated similarly.Tetranitromethane was found to cause changes in lactoferrin that went far beyond the mere nitration of a few tyrosine residues. Much of the tyrosine was destroyed and the same applied to tryptophan, part of which was converted to unidentified reaction products.
The Mechanism of Activation of Mutant β‐Galactosidase by Specific Antibodies
Authors: Melchers Fritz , Messer Walter 
Published: March 2005

Measurements of the rates of activation of mutant β‐galactosidases of Escherichia coli by specific antibodies to enzyme activity suggest that the rate‐limiting step of this activation reaction is the mono‐molecular reaction, with which a mutant enzyme population changes between two conformations. Four mutant β‐galactosidases produced by one group of mutants all have an activation energy for this monomolecular reaction of 23.2 kcal/mol. Mutant enzyme‐activating antibodies appear to change the equilibrium between a wild‐type‐enzyme and a mutant‐enzyme conformation to more active molecules by stabilizing the wild‐type conformation. Different antisera activate one mutant enzyme population to different specific activities, all below that of the wild‐type enzyme.Activating antibodies can be enriched from antibodies with specificities against determinants on the enzyme by passage of antiserum over Sepharose‐mutant enzyme immunosorbent columns.
Affinité comparée des sérums embryonnaires et maternels pour la corticostérone et la progestérone chez le rat
Authors: Savu Lia , Crépy Odette , Nunez Emmanuel , Jayle Max‐Fernand 
Published: March 2005

The binding parameters of corticosterone and progesterone have been measured comparatively in the blood sera from rat, embryos and pregnant rats, at the 19th day of pregnancy.

The level of corticosterone binding is significantly higher in the sera of foetal rats than in in the sera of pregnant rats. In contrast, the fixation of progesterone appears quantitatively similar in the embryo and in the pregnant rat.
Scatchard analysis shows that a single protein species, with high specific affinity for corticosterone is present in the foetal as well as in the pregnant serum. The association constants have similar values, i.e.Kass= 3 to 5 × 108 M‐1 at 25 ° C; these values are characteristic for the corticosterone‐binding globulin of the rat. However, the binding capacity [concentration of binding sites (n) × number of moles of binding protein (M)] is 2 to 3 times higher in the foetal serum. The results are interpreted as indicating that the higher levels of fixation of this steroid in the embryo, compared to the mother, are due to a quantitative rather than qualitative difference between their binding proteins.
The systems involved in the binding of progesterone are more complex in the embryo as well as in the pregnant rat. The values of the association constants and the results of competition and heat‐inactivation experiments suggest a probable contribution of the corticosterone binding globulin to these systems. However, the progesterone seems to bind mainly to one or several proteins with low affinity (Kass= 6 × 106 M‐1 at 25 °C) and great binding capacity (n×M= 215 × 10‐8/g of serum proteins). The foetal and pregnant rat sera appear to behave similarly in the process of progesterone binding.
Outer Membrane of Salmonella
Authors: Mühlradt Peter F. , Menzel Johannes , Golecki Jochen R. , Speth Volker 
Published: March 2005

The UDP‐galactose‐4‐epimeraseless strain 1195 of Salmonella typhimurium bears incomplete lipopolysaccharide, and only after addition of galactose to the growth medium it produces wild‐type lipopolysaccharide. It was demonstrated in a double‐label experiment that already existent, incomplete lipopolysaccharide is not completed after galactose addition but that only the newly synthesised lipopolysaccharide carries the wild‐type specificity.It was thus possible to locate newly synthesised lipopolysaccharide with specific anti‐wild‐type, ferritin‐conjugated antibodies in freeze‐etch preparations and ultra‐thin sections.With the freeze‐etch technique new lipopolysaccharide was found to appear in about 220 patches per cell, 30 s after 50 mM galactose had been added to the medium. After growth for 2–3 min under these conditions new lipopolysaccharide was found to be evenly spread over the entire cell surface.In ultra‐thin sections of plasmolysed cells 86% of the ferritin patches, indicating newly synthesised lipopolysaccharide, was found located over sites where the cytoplasmic and the outer membrane adhere to one another. It is discussed that new lipopolysaccharide emerges from these sites and that lateral movement of lipopolysaccharide in the outer membrane results in an even distribution on the bacterial cell surface.
Inactivation of α‐Chymotrypsin by a Bifunctional Reagent, 3,4‐Dihydro‐3,4‐dibromo‐6‐bromomethylcoumarin
Authors: Béchet Jean‐Jacques , Dupaix Alain , Yon Jeannine , Wakselman Michel , Robert Jean‐Claude , Vilkas Michel 
Published: March 2005

α‐Chymotrypsin is rapidly and irreversibly inactivated by 3,4‐dihydro‐3,4‐dibromo‐6‐bromo‐methylcoumarin (VII) at neutral pH. The inactivation is pH dependent between pH 5 and 7 and is delayed when the active site of the enzyme is protected by acetylation of the active serine‐195 or by binding of a competitive inhibitor.This dihydrocoumarin VII and its 6‐methyl analogue are substrates for α‐chymotrypsin, but when the ester bond in VII is broken during formation of the acyl‐enzyme, a reactive p‐hydroxybenzylbromide group is generated in situ within the active site. This newly formed group then alkylates one histidine residue (probably histidine‐57), as shown by amino acid analysis of the modified enzyme on acid hydrolysis. A model non‐enzymic reaction shows that the dihydrocoumarin VII is able to alkylate imidazole in aqueous solution at pH 7, yielding the N‐substituted imidazole, 3‐bromo‐6‐(imidazol‐1‐yl‐methyl)coumarin (XI).The modified enzyme has practically no activity against a specific substrate and seems no longer to have its intact active site. However its binding site is at least partly free for it is still able to bind proflavin.α‐Chymotrypsin is also inactivated by 6‐bromomethylcoumarin, the ester bond of which is stable, but the mode of inactivation and the properties of the modified enzyme are different from those found in the study with the dihydrocoumarin VII.
A Specific Ribonucleoprotein Fragment from Escherichia coli 30‐S Ribosomes
Authors: Székely Maria , Brimacombe Richard , Morgan Joan 
Published: March 2005

32P‐labelled ribosomal sub‐particles were hydrolysed with ribonuclease T1, under conditions which have previously been shown to yield a specific fragment of RNA · protein (“band III”), containing only four of the 21 30‐S ribosomal proteins. The hydrolysate was fractionated on a polyacrylamide gel using the methods already published, and gel fractions containing the specific RNA · protein fragment were subjected to electrophoresis on a second polyacrylamide gel, in the presence of sodium dodecylsulphate to dissociate RNA and protein. Two bands of [32P]RNA were reproducibly observed, containing about 320 and 360 nucleotides, respectively.
Two‐dimensional fingerprints of ribonuclease T1, hydrolysates from both these RNA species showed that the two fragments were almost identical. Oligonucleotides from the fingerprints were digested with pancreatic ribonuclease, and the secondary digestion products analysed and compared with the known sequence of 16‐S ribosomal RNA. Several characteristic oligonucleotides from regions D, E and P of the RNA were identified unambiguously. Other oligonucleotides were found which could have arisen from more than one part of the 16‐S RNA, but in each case one of the possibilities was in regions D, E, E′ or P.
It was concluded that the band III proteins are associated with these contiguous regions of the 16‐S RNA, i.e. near to its 3′‐end.
The Specificity of Yeast Fatty‐Acid Synthetase with Respect to the “Priming” Substrate
Authors: Pirson Wolfgang , Schuhmann Lieselotte , Lynen Feodor 
Published: March 2005

Yeast fatty acid synthetase catalyzes the synthesis of palmityl‐CoA and stearyl‐CoA from acetyl‐CoA and malonyl‐CoA. Acetyl‐CoA serves as “primer” of the reaction cycle. The specificity of the fatty acid synthetase with respect to the “priming” substrate was studied on the purified enzyme.

In its function as primer acetyl‐CoA was replaced by homologous saturated acyl‐CoA derivatives (butyryl‐CoA, capronyl‐CoA, etc.). In these experiments nonanoyl‐CoA and decanoyl‐CoA were good primers in low concentration range. This kind of fatty acid synthesis leads to normal end products.
At higher concentrations of priming substrate an inhibitory effect became apparent with all long‐chain acyl‐CoA derivatives. With decanoyl‐CoA the degree of inhibition was dependent on the concentration of malonyl‐CoA. This was interpreted as competition between decanoyl‐CoA and malonyl‐CoA.
The rate of synthesis with dl‐3‐hydroxydecanoyl‐CoA and Δ2‐decenoyl‐CoA as primer was 25–50 times slower than with decanoyl‐CoA. The rate‐limiting step was the transfer of the acyl residue from CoA to the enzyme. The saturated acids C12–C18 were isolated as end products of this synthesis.
Derivatives of decanoyl‐CoA with substitutions at positions more distant from the carboxyl group were not so strongly depressed in their priming activity, e.g.Δ3‐decenoyl‐CoA or 4‐oxo‐decanoyl‐CoA. In these cases the functional groups were retained during chain elongation. As reaction products unsaturated acids or oxo acids respectively were isolated.
The enzymatic reduction of the double bond (Δ3‐decenoyl‐CoA) or the oxo group (4‐oxo‐decanoyl‐CoA) could be demonstrated only as a slow side reaction.
The Anomalous Absorption of Tryptophan
Authors: ElÖDI Pal , Lakatos Susan 
Published: March 2005

The influence of polarizable organic molecules, e.g. organic acids and ionic detergents, on the light absorption of indole, tryptophan and its derivatives was studied. The interaction of these compounds with the chromophore, in addition to the red shift due to solvent perturbation effect, results in an “anomalous” blue shift in the 295 to 310‐nm region.
The nature of spectral changes depended on the character of the interacting solvent and solute molecules: a net positive electrostatic field induced red shift, whereas a negative one caused blue shift. The absorption changes increased with the size of nonpolar part of the polarizable molecule. The spectral changes can be assigned to the electrostatic attraction or repulsion of electrons of the indole nitrogen according to the electrostatic character of the polarizable molecule interacting with the chromophore.
The energy of indole chromophore‐polarizable molecule interaction was estimated from the data of thermal perturbation difference spectra. The calculated upper limit of this interaction energy was 0.04 eV, which suggests that in aqueous solution van der Waals interactions are formed between the chromophore and the compounds containing polarizable or charged groups.
The Effect of Temperature on Gel Formation in Pig‐Gastric Mucus
Authors: Snary David , Allen Adrian , Pain Roger H. 
Published: March 2005

The viscosity and gel properties of gastric mucus are due to a mucoprotein of protein content of 18% and molecular weight 2 × 106.
At 25°C the reduced specific viscosity of this mucoprotein increases with increasing concentration until the mucoprotein undergoes a reversible sol‐gel transition at concentrations above 4 mg · ml−1.
For a given concentration of mucoprotein the viscosity increases markedly with temperature and at 45°C the threshold concentration for reversible gel formation is reduced to 2 mg · ml−1.
The intrinsic viscosity corrected for zero shear, the shear dependence of the viscosity, and the sedimentation properties of the mucoprotein as a function of temperature have been measured.
These experiments show that the increase of viscosity with temperature is due to increased intermolecular interaction rather than structural changes in the molecule.
The temperature dependence is characteristic of hydrophobic interactions and the results are interpreted to show that the mechanism of gel formation in gastric mucus involves micelle junctions.
The Conformational Transitions in Yeast tRNAPhe as Studied with tRNAPhe Fragments
Authors: Riesner Detlev , Maass Günter , Thiebe Rainer , Philippsen Peter , Zachau Hans G. 
Published: March 2005

Conformational transitions of the anticodon branch, the acceptor branch and the tertiary structure of yeast tRNAPhe were investigated using tRNAPhe fragments. Thermodynamic and kinetic methods were employed using the optical absorbance of the fragments and the fluorescence of the Y‐base. Related results of partial nuclease digestions on tRNAPhe fragments are also reported.

Hypochromicity, reaction enthalpy and rate constants of helix‐coil transitions of the anti codon branch agree with the values expected from the cloverleaf model. The dissociation of the branch is accompanied by a quenching of the fluorescence of the Y‐base.
In some isolated fragments the anticodon branch is more stable than in the intact tRNA. The destabilization in the intact tRNA is attributed to electrostatic effects.
The anticodon half Phe 21–57, at low concentrations and temperatures, assumes a structure which is different from a segment of the cloverleaf model. A transition from this structure to the one of a segment of the cloverleaf is observed with increasing temperature. At higher fragment concentrations and in the presence of Mg2+ dimers are formed.
Bimolecular recombinations are observed between fragments Phe 1–18 and Phe 21–76 reconstituting the acceptor and dihydrouridine stems, and between Phe 1–18 and Phe 38–76 where only the base pairs of the acceptor stem are newly formed. The mechanism of the recombination and the relationship of the fragment combination to the intact tRNA are discussed.
The thermodynamic properties of the fragment Phe 1‐69/70 are very similar to those of the intact tRNA. It can be concluded that the tertiary structure is essentially unchanged anti that the terminal nucleotides 70–76 are not directly involved in its formation.
The thermal denaturation process of intact tRNAPhe can now be interpreted as follows: at first the tertiary structure is converted to a cloverleaf‐like structure; this process is followed by the melting of the acceptor and anticodon stems; finally the ribosylthymine and dihydrouridine stems dissociate independently.
Purification and Molecular Properties of an Unspecific Carboxylesterase (E1) from Rat‐Liver Microsomes
Authors: Arndt Rüdiger , Heymann Eberhard , Junge Wolfgang , Krisch Klaus , Hollandt Heinrich 
Published: March 2005

The isolation of an unspecific carboxylesterase from rat liver microsomes is reported. The purification is 66‐fold at a yield of about 20%. The enzyme is one of 5 esterase/amidase variants recently separated and corresponds to the fraction designated E1.
The enzyme is homogeneous in disc electrophoresis, dodecylsulfate‐polyacrylamide gel electrophoresis and in analytical ultracentrifugation, but exhibits slight heterogeneity in isoelectric focusing.
Its amino acid composition is presented.
The molecular weight of rat liver esterase E1 is 177000 (equilibrium sedimentation), the subunit weight 61 500 (dodecylsulfate disc electrophoresis), and the equivalent weight 66000 (titration with bis[p‐nitrophenyl]phosphate). After cross‐linking of the enzyme by dimethyl suberimidate and dodecylsulfate‐polyacrylamide gel electrophoresis three principal bands are observed. Therefore, a trimeric structure in which each subunit possesses one active site is proposed.
Catalytic Properties of an Unspecific Carboxylesterase (E1) from Rat‐Liver Microsomes
Authors: Arndt Rüdiger , Krisch Klaus 
Published: March 2005

In a study on the influence of the alkyl and acyl chain length of aliphatic esters on kinetic parameters of rat liver carboxylesterase (E1) it was found that V is mainly governed by the length of the acyl residue, whereas on elongation of the alcohol group pKm is increased.
By halogen substitution of ethyl acetate in the α‐position of the acetate, the pKm values are increased in the order F < Cl < Br < I.
Besides aliphatic esters, also aromatic ones, such as l‐tyrosine ester, procaine, p‐nitrophenyl acetate and, as an aromatic amide, butacetoloid (hostacaine®) are hydrolyzed.
The pH‐profile was found to depend on the nature and concentration of the substrate.
In contrast to the results obtained with the purified enzyme, no substrate inhibition and a different pH‐profile were observed on incubation of rat liver microsomes with methyl butyrate.
Hydrolysis of Amino‐Acid Esters by Pig‐Pancreatic Kallikrein
Authors: Fiedler Franz , Leysath Gisela , Werle Eugen 
Published: March 2005

Initial velocities of the hydrolysis of α‐N‐acylated esters of 14 different amino acids catalyzed by pig pancreatic kallikrein were determined. Control experiments with kallikrein partially inactivated by controlled treatment with diisopropylphosphorofluoridate demonstrated the enzymic homogeneity of the preparation and the ability of kallikrein to hydrolyse (at decreasing rates) esters of basic, aromatic and neutral aliphatic amino acids, though being predominantly specific for l‐arginine esters. The controversial literature on the substrate specificity of the enzyme has its origin not in the molecular heterogeneity of kallikrein, but in a contamination with other enzymes. Kinetic constants for the kallikrein‐catalyzed hydrolysis of several of the esters have been determined and compared to those of trypsin and clostripain. The preference of kallikrein for an arginine over a lysine ester is still higher than with the latter enzyme. In the α‐N‐benzoylated as well as in the α‐N‐tosylated series, esters of l‐arginine with different alcohol moieties exhibit not only widely different Km, but also Kcat values. The latter parallel the rates of the hydroxyl‐ion‐catalyzed hydrolyses of the α‐N‐benzoyl‐l‐arginine esters which have also been determined.Syntheses of α‐N‐benzoyl‐l‐lysine methyl ester hydrochloride and of α‐N‐benzoyl‐l‐ornithine methyl ester hydrochloride are described. A value for the rate constant of the hydroxyl‐ion‐catalyzed lactamization of the ornithine ester has been obtained.
The Effect of Fluoride on the Spectral and Catalytic Properties of Three Copper‐Containing Oxidases
Authors: BrÄNDÉN Rolf , MalmstrÖM Bo G. , Vänngård Tore 
Published: March 2005

The optical spectra of fungal laccase, lacquer tree laccase and ceruloplasmin are changed in a similar way when F– is added to the proteins at pH 5.5.
A change around 320 nm is associated with the binding of F– to type 2 Cu2+ of the proteins as seen from a correlation with electron paramagnetic resonance spectra.
The rate of binding of F– to fungal laccase is first order with respect to uncomplexed enzyme and independent of the F– concentration, indicating that an intramolecular process is rate limiting. The stability constant of the enzyme · F– complex is greater than 10 μM‐1.
Fungal laccase is almost completely inhibited when 1 equivalent of F– is bound to the type 2 Cu2+. Presence of substrate gradually decreases the inhibition, due to the release of F– from a reduced form of the enzyme.
The Mechanism of Action of Methionyl‐tRNA Synthetase
Authors: Blanquet Sylvain , Petrissant Guy , Waller Jean‐Pierre 
Published: March 2005

The interactions of three methionine‐specific tRNAs (initiator tRNAfMet from rabbit liver, cross‐linked tRNAfMet and untreated tRNAfMet from Escherichia coli) with methionyl‐tRNA synthetase from E. coli were examined. Binding parameters determined by fluorescence spectroscopy were compared with kinetic parameters derived from the aminoacylation reaction performed under comparable experimental conditions.Cross‐linked tRNAfMet and untreated tRNAfMet are virtually indistinguishable on the bases of their kinetic parameters in the aminoacylation reaction, but can be differentiated by fluorescence measurements. The lower level of enzyme fluorescence quenching elicited by the cross‐linked species must be related to a structural modification which has no significant repercussion on its performance in the aminoacylation reaction.The initiator tRNA from rabbit liver differs from that of E. coli in several respects. Its binding parameters, measured by fluorescence, are characterized by a two‐fold higher Ka, a two‐fold lower maximal level of enzyme fluorescence quenching and a less pronounced sensitivity to the addition of KCl. The lower sensitivity to KCl is also reflected in the kinetic parameters measured in the aminoacylation reaction.Several unspecific tRNAs which are not aminoacylated by the enzyme under standard conditions are able to interact with it to significant extents, as assessed by fluorescence measurements. These interactions are characterized by weak affinities and small effects on protein fluorescence, comparable in magnitude to those observed when specific tRNAs interact with the enzyme in the absence of magnesium ions.