bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thiol Function and Tertiary Structure of RNA Polymerase of Escherichia coli
Authors: Nicholson Bruce H. , King Andrew M. Q. 
Published: March 2005

RNA polymerase requires thiol compounds to maintain optimum activity during isolation, although it is not a thiol enzyme.
Total thiols have been determined for RNA core polymerase by reaction with p‐chloro‐mercuribenzoate, 2‐chloromercuri‐4‐nitrophenol and 5,5′‐dithio‐bis(2‐nitrobenzoic acid). Their distribution between the subunits has been determined as ααββ' = 4.4.8.8.
An arbitrary division of the reaction rate profile shows that of the 18 thiols reacting with 5,5′‐dithio‐bis(2‐nitrobenzoec acid) in 18 h, over 10 react in 1% of this time. Most of these are buried thiols since only 2 or 3 react with N‐[p‐(‐methyl‐2‐benzthiazole)phenyl]maleimide.
Enzyme activity is lost gradually on titration with 5,5′‐dithio‐bis(2‐nitrobenzoic acid), but rapidly with p‐chloromercuribenzoate until 10–12 thiols have reacted per mole; polymerase then undergoes a large reversible change in activity, although with 5,5′‐dithiobis(2‐nitrobenzoic acid) reversibility is slow and depends on the groups/mole that have reacted.
This inactivation by 5,5′‐dithio‐bis(2‐nitrobenzoic acid) may be prevented by pre‐initiating the enzyme which causes the reaction to stop at 11 thiols/mole, leaving 60–70% of the original activity. DNA on its own does not produce this resistance, which is proportional to the amount of initiated complex present.
Measurements with fluorescent probes show that considerable destabilisation of the tertiary structure occurs on reaction with p‐chloromercuribenzoate. Consideration of this together with the known subunit distribution of thiols strongly suggests that DNA acts by stabilising the structure rather than by direct steric hindrance.
Macromolecular Structure of Lipopolysaccharides from Gram‐Negative Bacteria
Authors: Hannecart‐Pokorni Eleonora , Dekegel Daniel , Depuydt Freddy 
Published: March 2005

The cell wall lipopolysaccharide constituent of Shigella flexneri, Salmonella typhimurium and Salmonella minnesota appears in the electron microscope as long slender filaments having a molecular weight of several millions. Such lipopolysaccharides are constituted of subunits held together by low‐energy bonds of chelating and hydrophobic nature due to the lipid part of the molecule. Physicochemical studies gave molecular weights in the range of 18000 to 25000 for the subunits of S strains depending upon the bacterial species, and 10000 in the case of the Salmonella minnesota R mutants. The subunits seem to be long ellipsoids of revolution made up of a lipid part and two polysaccharide side chains.Studies of the dissociation and the reassociation of the lipopolysaccharide subunits showed the existence of a cylindrical intermediate “unit structure” having a molecular weight of about 5·105. The arrangement of the subunits in that unit structure seems to be of a micellar type, the lipids being located in the central part of this structure. This latter model is compatible with a skew arrangement of unit structures into the larger polymer molecules.
Pyrimidine‐Ribonucleotide Pools and Their Turnover in Phage T4‐Infected Escherichia coli Cells
Authors: Fast Robert , Sköld Ola 
Published: March 2005

Exogenous uracil was shown to be taken up efficiently into ribonucleotide pools at all times during the cycle of T4 phage infection, in which stable host RNA synthesis is known to be turned off. This uptake was shown to be effected by ribonucleotide pool expansion and a net increase in mRNA content after phage infection. By comparing different phage mutants, the draining of ribonucleotide pools into DNA synthesis by ribotide reduction, was also found to be instrumental for pool labeling from exogenous uracil. The UDPG pool expanded as much as 3‐fold during DNA negative mutant infection, presumably as a result of absent DNA glucosylation.From measurements of specific activities of nucleotides by double labeling and thin‐layer chromatography, it was concluded that the incorporation into RNA of a radioactive, exogenous base can be reliably used for following T4 phage transcription.
Properties of Arginine Kinase from Drosophila melanogaster
Authors: Wallimann Theo , Eppenberger Hans M. 
Published: March 2005

1. Starch‐gel electrophoresis of extracts of several strains of the genus Drosophila reveals neither allelic variants nor isoenzymes of arginine kinase. Arginine kinase of the drosophilid Zaprionus vittiger migrates differently.2. Arginine kinase is non‐uniformly distributed in tissues of Drosophila melanogaster. The activity in muscle tissue represents about 70% of the total activity. A characteristic fluctuation of the enzyme activity during the development from the egg to the adult stage can be observed.3. Purification of arginine kinase of Drosophila melanogaster has been achieved. Several physico‐chemical properties of the enzyme have been determined. The molecular weight in the range of 40 000 is comparable to other arthropod arginine kinases and suggests that the active enzyme is a monomer.
Purification and Properties of Pig‐Heart Hexokinase
Authors: Easterby John S. , O'Brien Michael J. 
Published: March 2005

Hexokinase has been purified from pig heart to a specific activity of 80 units/mg.The enzyme has an s20 of 5.11 ± 0.15 S and a molecular weight of 97000. Dodecylsulphate‐polyacrylamide electrophoresis and maleylation of the enzyme each suggest that it contains a single polypeptide chain. Amino‐acid analysis reveals similarities between the compositions of heart and brain hexokinases. Steady‐state kinetic investigations at sub‐optimal substrate concentrations are consistent with a mechanism in which at least one of the substrates is in equilibrium with its enzyme · substrate complex.
Carbon‐Dioxide Concentration and the Distribution of Monocarboxylate and H+ Ions between Intracellular and Extracellular Spaces of Hemoglobin‐Free Perfused Rat Liver
Authors: Sies Helmut , Noack Gerhild , Halder Karl‐Heinz 
Published: March 2005

H+ movement and monocarboxylate‐monocarboxylate exchange accompanying monocarboxylate gradient equilibration in hemoglobin‐free perfused rat liver have been studied in an isopH system using different buffer systems.1. At 5–10 mM concentrations of added monocarboxylate, there is an approximately linear dependence of net H+ uptake by the liver on added monocarboxylate. Net H+ uptake at 10 mM additions is 1.5–1.6 μmol per gram liver from aromatic weak acids like benzoate or salicylate, and 0.6–0.8 μmol per gram liver for aliphatic weak acids like acetate, lactate, pyruvate, and also 5,5‐dimethyloxazolidine‐2,4‐dione.2. Practically no net H+ movement is observed with dicarboxylates like phthalate or succinate and also not with choline chloride.3. Monocarboxylate‐monocarboxylate exchange is demonstrated by preloading the hepatocytes with benzoate, lactate and pyruvate, and observation of their release upon addition of other monocarboxylates.4. Net H+ uptake increases as total CO2 in perfusate is decreased at constant extracellular pH, in accordance with an intracellular acidification by the CO2 system. This is supported by measurement of distribution of 5,5‐dimethyloxazolidine‐2,4‐dione, and by monocarboxylate release from the cells during increases of CO2 concentration.5. Intracellular pH poise is low when bicarbonate buffer is replaced by zwitterionic buffers like N‐2‐hydroxyethylpiperazine‐N′‐ethane sulfonic acid or morpholinopropane sulfonic acid, resulting in calculated intracellular alkalinization of up to 0.5 pH unit. 5,5‐Dimethyloxazolidine‐2,4‐dione as a permeant buffer system is suggested as a more suitable replacement of bicarbonate buffer than the zwitterionic buffers with respect to intracellular pH poise.
The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)
Authors: Vincent Jean‐Pierre , Lazdunski Michel 
Published: March 2005

1. The association constant, Ka, of the 1:1 complex formed between α‐chymotrypsin and the pancreatic trypsin inhibitor is 110 μM−1 at pH 8.0 and 25 °C. The second‐order rate constant for the association, ka, is 110 mM−1 s−1 and the first‐order rate constant for the dissociation, kd, is 10−3 s−1 under the same conditions. Thermodynamic parameters for complex formation at 25 °C, pH 8.0 are ΔGa0= ‐11.0 kcal×mol−1, ΔHa0= 3 kcal×mol−1 and ΔSa0= 48 cal. · mol−1· K−1.2. Temperature and pH‐dependences of the rate constants ka and kd have been studied.3. The difference in stability between the α‐chymotrypsin · inhibitor complex and the trypsin · inhibitor complex (Ka= 16 pM−1, ΔGa0=–18.1 kcal×mol−1) is due essentially to differences of kd values. The dissociation of the α‐chymotrypsin · inhibitor complex is 1.5×104 times faster than that of the trypsin · inhibitor complex. Differences in Ka and kd values are discussed in terms of differences in stabilizing interactions.4. The Cys14‐Cys38 disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the α‐chymotrypsin · inhibitor complex. Reduction of the Cys14‐Cys38 bridge in the free inhibitor does not prevent association with α‐chymotrypsin. Values of ka and kd for the formation of the 1:1 α‐chymotrypsin · reduced‐inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin · pancreatic‐inhibitor complex. In that case, reduction of Cys14‐Cys38 bridge considerably decreases the stability of the complex formed with trypsin; Ka is decreased by a factor of 3×104 and kd is increased by a factor of 8.6×103.
Sites of Synthesis of Chloroplast‐Membrane Proteins
Authors: Apel Klaus , Schweiger Hans‐Georg 
Published: March 2005

The protein synthesis of isolated chloroplasts of Acetabularia has been measured by the in‐corporation of labeled amino acids into the trichloroacetic‐acid‐insoluble fraction as well as into individual membrane fractions.Chloramphenicol inhibited the incorporation into the trichloroacetic‐acid‐insoluble material only up to 60%, as did cycloheximide. The effect of the two antibiotics on the incorporation into the trichloroacetic‐acid‐insoluble material of isolated chloroplasts is additive.By the treatment of chloroplasts with EDTA most of the originally membrane‐bound proteins were dissolved and could be separated quantitatively from EDTA‐insoluble material. Only three protein components could be detected in the membrane fragments. Two of these membrane proteins were synthesized in isolated chloroplasts. The synthesis of one of these two proteins is inhibited completely by cycloheximide, while the labeling of the other protein has scarcely been affected. The synthesis of the cycloheximide‐insensitive protein is almost completely blocked by chloramphenicol, but the cycloheximide‐sensitive protein still has been labeled distinctly in the presence of chloramphenicol. It is concluded that these chloroplast proteins are synthesized on two different groups of ribosomes, both of which are present in the isolated chloroplasts. The presence of 80‐S ribosomes in this chloroplast preparation has been indicated by finding 26‐S rRNA. Thus it is probable that the cycloheximide‐sensitive particles in the chloroplast preparation are identical with 80‐S ribosomes.While in isolated‐chloroplasts only two membrane proteins have been synthesized, all three EDTA‐insoluble membrane proteins have been labeled in whole cells. It is concluded that the synthesis of the third protein takes place on a ribosome group in the cytosol which is active only in the intact cell.
The Degradation of CoA: Subcellular Localization and Kinetic Properties of CoA‐and Dephospho‐CoA Pyrophosphatase
Authors: Skrede Sverre 
Published: March 2005

1. An enzyme degrading dephospho‐CoA with 5′‐AMP as the reaction product is present in the plasma membranes of rat liver. CoA is degraded at less than 10% of the rate of dephospho‐CoA.2. The presence of plasma membranes in nuclear and microsomal fractions, which also contain significant amounts of the plasma‐membrane marker enzymes, probably explains the presence of dephospho‐CoA pyrophosphatase in these fractions. The enzyme is different from inorganic pyrophosphatase present in nuclei and microsomes with respect to pH optimum and sensitivity to inhibitors.3. Dephospho‐CoA pyrophosphatase is inactivated by dialysis against EDTA, and is partially reactivated by Mn2+ and a number of other divalent cations.4. The pyrophosphatase degrading dephospho‐CoA and CoA is probably identical with nucleotide pyrophosphatase from plasma membranes. The degradation of dephospho‐CoA is inhibited competitively by nucleotides with a pyrophosphate bond; most strongly by NADH, which has the highest affinity for nucleotide pyrophosphatase.5. Competitive inhibitors of dephospho‐CoA degradation are less efficient when a ribose 3′‐phosphate is present in the nucleotide. Km for dephospho‐CoA is 0.02 mM, for CoA about 0.3 mM. These results indicate that the 3′‐phosphate group is a sterical hindrance for the access of the nucleotides to the active site. V for dephospho‐CoA in plasma membranes was about 200 nmol×mg protein−1×min−1, but for CoA one order of magnitude less.6. Also nucleotide pyrophosphatase from Crotalus adamanteus degraded CoA at a much lower rate than dephospho‐CoA (below 1%).
Kinetic Analysis of a Rat‐Liver Nucleotidase Activated by Deoxyribonucleic‐Acid Constituents
Authors: Fritzson Per 
Published: March 2005

A nucleotidase present in rat liver cytosol which hydrolyzes certain 3′‐ and 5′‐nucleotides, is subject to allosteric activation by dIno, dGuo, dThd, dUrd, and the corresponding 5′‐deoxy‐ribonucleotides. A systematic study of enzyme activation as a function of effector concentration was carried out with Urd‐3′‐P or [14C]dThd‐3′‐P as substrate. It was found that optimal effector concentration was 6–8 mM for the nucleosides, 4 mM for dThd‐5′‐P, and 1–2 mM for the other 5′‐deoxyribonucleotides. Deoxyinosine was a stronger activator than any of the other effectors and afforded a 3.3‐fold increase in the rate of Urd‐3′‐P hydrolysis at optimal concentration. At 1 mM concentration the enzyme was stimulated 2.3 times by dIno and, furthermore, by dGuo dGuo‐5′‐P, dThd‐5′‐P, dUrd‐5′‐P, and dIno‐5′‐P in decreasing order of effectiveness. The di‐ and triphosphates of dThd were more stimulatory than the monophosphate at concentrations above 3 mM. The p‐nitrophenyl ester of dThd‐5′‐P showed stimulation similar to that of the unesterified compound.A kinetic enzyme model was developed which explains all the observed relations between enzyme activity and effector concentration. The parameters of the model were calculated by means of a programmed electronic computer and showed that the apparent dissociation constants for binding of dIno and the 5′‐monophosphates of dIno, dGuo, and dUrd to the regulatory site were about 1.7 mM. The other effectors had higher dissociation constants. The apparent Km values of the 5′‐deoxyribonucleotides, calculated by means of the computer, were about 0.2 mM. This value is similar to the Km values of the 3′‐nucleotides.The stimulation of enzyme activity is due to an increase in V without a detectable change in the apparent Km, a mechanism which is less common for regulatory enzymes.
The Reactivity of Escherichia coli Ribosomal Sulphydryl Groups with 5,5′‐Dithiobis(2‐nitrobenzoic Acid)
Authors: Johnson Peter M. , Walker Ian O. 
Published: March 2005

A study has been made of the reactivity of the sulphydryl groups of Escherichia coli ribosomes with 5,5′‐dithiobis(2‐nitrobenzoic acid). Our results show that all the sulphydryl groups of the ribosomal proteins will react with this sulphydryl‐specific reagent.Measurements of the rate of reaction provide evidence for conformational changes, independent of the association –dissociation reaction. The presence of 100 mM K+ or Na+ will markedly reduce the reactivity of the sulphydryl groups of 70‐S ribosomes or the ribosomal subunits. This conformational change has been interpreted in terms of a “tightening” of the ribosomal structure by the binding of monovalent cations.A similar conformational change was observed on decreasing the temperature of the reaction mixture. The difference in the reaction rate was more than can be explained by the temperature dependence of the kinetics of the simple reaction, investigated using a model sulphydryl compound, 2‐mercaptoethanol.
Comparative Study of Photophosphorylation Coupling Factor · Ligand Complex by Circular Dichroism and Chemical Isolation
Authors: Girault Guy , Galmiche Jean‐Michel , Michel‐Villaz Michel , Thiery Jean 
Published: March 2005

1. The interaction between the protein coupling of photophosphorylation and its substrate ADP is studied comparatively by two methods: circular dichroism and chemical isolation of the ligand · enzyme complex.2. Experiments with 14C‐labelled ADP indicate that the amount of bound nucleotide is proportional to a variation of the circular dichroism spectrum around 260 nm. When coupling factor is saturated with ADP, two moles of ADP are bound per mole of coupling factor.3. The fixation of ADP on coupling factor is inhibited by the presence, in the incubation medium, of phosphate (Pi), pyrophosphate (PPi) or tripolyphosphate ions. The incubation of coupling factor with [32P]PPi shows that PPi inhibition is due to its fixation on the coupling factor. When coupling factor is saturated, two moles of PPi are bound per mole of coupling factor.
Experimental Evidence for a Hydrophobic Active Center of Glutamic‐Aspartic Transaminase
Authors: Dimitropoulos Constantin G. , Oikonomakos Nikolaos G. , Karni‐Katsadima Irene A. , Kalogerakos Theodore G. , Evangelopoulos Athanasios E. 
Published: March 2005

1. It has been found that fluorescein dimercuric acetate acts as a potential inhibitor of transaminase. Although the holoenzyme was inhibited also by fluorescein isothiocyanate the latter fluorochrome was found to be a rather specific inhibitor of the apoenzyme. The interactions involved are fast, the induced effect reaching its maximum value in less than 1 min.2. The isolated fluorescein dimercuric acetate or fluorescein isothiocyanate and transaminase or apotransaminase complexes suggest that the fluorochromes reacts specifically with two active sulfhydryl or amino groups of the enzyme, respectively.3. The fluorescence intensity of the fluorochromes is substantially quenched during their interaction with transaminase and apotransaminase. A red shift in the absorption spectrum maximum from 498 nm to 512nm was also observed when fluorescein dimercuric acetate solutions were titrated with transaminase or apotransaminase. The recorded shift for fluorescein isothiocyanate in the fluorescein‐isothiocyanate · apotransaminase complex was from 493 nm to 502 nm, respectively. In contrast to the previous spectral changes a blue shift in the spectrum maximum from 498 nm to 478 nm was recorded during the stoichiometric interaction of fluorescein dimercuric acetate with cysteine.4. Comparative studies concerning the reactivity of fluorescein dimercuric acetate or fluorescein isothiocyanate with transaminase, apotransaminase and cysteine or lysine suggest that the alterations observed in the spectral and fluorescence properties of the fluorochromes during their interaction with transaminase and apotransaminase arose from their complexation with specific sulfhydryl groups of the enzyme. These groups should be located in a critical hydrophobic area of the transaminase molecule since their interaction abolishes the catalytic activity of the enzyme and alters the spectral and fluorescence properties of the fluorochromes in the same way as low polarity solvents. These sulfhydryl and amino groups might also play a functional role since they interact faster with fluorescein dimercuric acetate and fluorescein isothiocyanate than the sulfhydryl group of cysteine or the amino group of lysine.5. Quantitative parameters of the data obtained by conventional techniques and stopped‐flow spectroscopy during the interaction of the enzyme with the fluorochromes are also given.
Specific Modification of Arginyl‐Transfer Ribonucleic‐Acid Synthetase from Bacillus stearothermophilus as a Result of Substrate Binding
Authors: Parfait Raymond 
Published: March 2005

The association between arginyl‐tRNA synthetase from Bacillus stearothermophilus and tRNAArg was studied. ARP does not change the equilibrium of this association. The Km values for homologous tRNA and tRNA from Escherichia coli, and the inhibition constant (Ki) for periodate‐oxidized tRNA were determined.The synergistic protection of arginyl‐tRNA synthetase against heat inactivation, induced by tRNAArg, ATP and arginine, occurs in two discrete steps, the first being observed when the enzyme is incubated in the presence of tRNAArg and ATP, the second in the additional presence of arginine. With periodate‐oxidized tRNA, only the first step is observed.Arginyl‐tRNA synthetase dimerizes in the presence of tRNA species which can be aminoacylated by the enzyme. An effect of ATP on the kinetic parameters of enzyme‐tRNA association was indicated.Fluorescence measurements show that one molecule of tRNAArg is bound per molecule of enzyme monomer (Mr 78000).It is concluded from these and previous results, that an intermediary complex composed of the enzyme dimer, to which two molecules of each substrate (tRNAArg, ATP, arginine) have been bound in an ordered sequence, forms prior to the aminoacylation reaction catalysed by the enzyme.
Cell Envelope and Shape of Escherichia coli K12
Authors: Henning Ulf , Höhn Barbara , Sonntag Ingeborg 
Published: March 2005

Rod‐shaped “ghosts” can be isolated from Escherichia coli (and a number of other gram‐negative organisms) which are devoid of murein, are surrounded by a unit membrane and have lost all intracellular material except for some internal membrane which is derived from the cytoplasmic membrane. Ghosts consist of about 25% phospholipid, 25–30% lipopolysaccharide and 45–50% protein.Dodecylsulfate‐gel electrophoresis resolves the protein into four main bands with the apparent molecular weights: I, 40000, II, 28000, III, 18000 and IV, 10000. The proteins of all four bands are predominantly located in the outer membrane of the cell wall. Band IV has been identified as the lipoprotein, part of which is bound covalently to murein [1]. Band II very likely represents a homogeneous polypeptide chain and is derived by the action of trypsin on ghosts from a protein II* which has a molecular weight of about 40000. The molar ratio of polypeptides I, II* and IV in ghosts is about 1:1:8–10. Protein of band III is present in smaller amounts which, in contrast to I, II* and IV, were found to vary from preparation to preparation.Proteins I, II* and IV comprise 7–10% of the total cellular protein and they are most likely arranged in the outer membrane of the cell envelope as repeating subunits. Several lines of evidence indicate that proteins of all four bands are also present in the cytoplasmic membrane, although in considerably lower quantities than in the outer membrane. The synthesis of these cell wall proteins does not require DNA or murein synthesis.The phospholipid in ghosts is not required for their macroscopic structural integrity or for the unit membrane appearance of either of their membrane systems. A phospholipid bilayer is therefore not the basic structure of the ghost membranes.