bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy Transduction in Mitochondrial Fragments
Authors: Azzi Angelo , Fabbro Antonio , Santato Mario , Gherardini Pier Luigi
Published: March 2005

Atebrin and acridine orange fluorescence and absorbance characteristics are sensitive to their environment.

Anions, such as iodide, nitrate or chloride strongly decrease acridines fluorescence, while sulfate or acetate are much less effective.

Energization by succinate or ATP of rat liver mitochondrial fragments produces a fluorescence decrease of atebrin, only in the presence of iodide, nitrate or chloride. In the presence of sulfate or acetate or in the absence of anions, no changes are detected.

Binding changes of acridines are also observed in association with energy conservation in mitochondrial fragments in the presence of iodide, nitrate or chloride.

These data are consistent with a model in which the membrane of mitochondrial fragments, becoming more positive than the medium when energized, creates local electrolyte concentration changes affecting fluorescence, absorbance and binding properties of acridine dyes.
Mechanism of Microbial Transformation of Cholesterol into Coprostanol
Authors: Björkhem Ingemar , Gustafsson Jan‐Åke
Published: March 2005

The conversion of cholesterol into coprostanol by cecal contents from rats under anaerobic conditions was studied using deuterated water, [4β‐3H,4‐14C]‐ and [3α‐3H,4‐14C]cholesterol. On the basis of the following experimental findings, it was concluded that the conversion of cholesterol into coprostanol proceeds to at least 50% by means of the intermediate formation of Δ4‐cholestenone.

Conversion of [3α‐3H,4‐14C]cholesterol into coprostanol occurred with loss of 50% of the tritium. The tritium retained in coprostanol was located in the C‐3α position. However, part of the tritium retained might have been introduced in a reductive step following an initial oxidative step. Evidence for this possibility was provided by the finding that coprostanol formed from [4‐14C]cholesterol in the presence of [3α‐3H]β‐sitosterol contained significant amounts of tritium in the C‐3α position.

After conversion of part of [3α‐3H,4‐14C]cholesterol into coprostanol, unchanged cholesterol had a higher 3H/14C ratio than the cholesterol added to the incubation mixture, indicating that oxidation of the 3β‐hydroxyl group is at least partly rate limiting in the over‐all conversion of cholesterol into coprostanol.

After incubation of cholesterol in the presence of deuterated water, the coprostanol isolated had a deuterium content of about 1.4 atoms at C‐2, C‐3 and C‐4, about 0.5 atoms at C‐5 and about 0.1 atoms at C‐6, indicating extensive oxido‐reduction at C‐3 during or after conversion of cholesterol into coprostanol.

Coprostanol isolated after incubation of [4β‐3H,4‐14C]cholesterol retained 60% of the tritium. The main part of this tritium had been transferred to the C‐6 position, showing that the conversion of cholesterol into coprostanol involves isomerization of the Δ5 double bond to a Δ4 double bond.

Incubation of Δ4‐[4‐14C]cholestenone resulted in efficient formation of coprostanol.
Cold Inactivation of l‐Threonine Deaminase from Rhodospirillum rubrum
Authors: Feldberg Ross S. , Datta Prasanta
Published: March 2005

Threonine deaminase from Rhodospirillum rubrum is subject to reversible cold inactivation at low protein concentrations. The inactivation appears to involve a dissociation of the native tetrameric protein to dimers. The standard free energy of dissociation at 0 °C was + 7.8 kcal/mole, while ΔS was—3.2 kcal/mole and ΔS was—41 entropy units. These values suggest that hydrophobic interactions are important in the maintenance of the tetrameric structure.

The labilization of the protein by low concentrations of urea and the protection by high salt concentrations also indicate the importance of hydrophobic bonds in the protein. However, the effects of pH and low salt concentrations on the cold inactivation suggest that ionic bonds do play some role in subunit interactions.

The normal allosteric modifier for threonine deaminase, l‐isoleucine, protected the enzyme against cold inactivation. Although the R. rubrum enzyme is virtually insensitive to isoleucine inhibition, it is clear that this ligand can induce profound changes in the protein structure.
Bovine Luteinizing Hormone Study of the Primary Structure around the Carbohydrate Attachment Sites of the Luteinizing Hormone α‐Subunit
Authors: Maghuin‐Rogister Guy C. , Hennen Georges P.
Published: March 2005

The α‐subunit of bovine luteinizing hormone is electrophoretically heterogeneous. Two subfractions of this subunit, designated LHαA and LHαB, were isolated and found homogeneous by electrophoresis.

Bovine LHαA was cleaved by both cyanogen bromide and trypsin. Two distinct groups of homologous glycopetides were defined. The first group corresponds to the carboxy‐terminal portion of the subunit. The sequence accounting for the major peptide population is the following: Gly‐Asx‐Val‐Arg‐GlxVal‐Glx‐Asx‐HisSer‐ Thr‐Glx‐Cys‐His‐SerHis‐Cys‐Thr‐Cys‐Tyr‐Tyr‐His‐Lys‐Ser‐ COOH. Ambiguities in the amino‐terminal sequence are observed at the fifth, eighth and thirteenth position.

Another population of peptides lacks three (or four) amino acids at the carboxy‐terminal end.

The second group of glycopeptides corresponds to an internal portion of the subunit. The most abundant peptide population exhibits the following sequence: Asx‐Thr‐Thr‐Ser‐Ala‐Glx‐Cys‐Ile‐Cys‐Val‐Ala‐Lys‐COOH. A variant population exhibits the sequence Asx‐Ile‐Thr‐Ser‐Ala‐.

Two sites are proposed for the attachment of the polysaccharide prosthetic groups, i.e. the tripeptide Asx‐HisSer‐Thr and the tripeptide Asx‐IleThr‐Thr, corresponding to the carboxy‐terminal portion and to the inner part of the subunit, respectively.

Within both groups of glycopeptides, a relationship was observed between the type of residue × occupying the α position at the carbonyl side of Asx in the tripeptide sequence Asx‐X‐Thr and the composition of the polysaccharide side chain.

Our data document the degree of homology existing between LHα and the common type of subunit present in the thyroid‐stimulating hormone molecule (i.e. the α‐subunit).
Espèces acétylcholinestérasiques globulaires et allongées des organes électriques de poissons
Authors: Massoulié Jean , Rieger François , Bon Suzanne
Published: March 2005

In the present communication we show that species A, C, D of both Torpedo and Electric eel acetylcholinesterase are totally excluded from Sephadex G‐200 and Biogel P‐300 molecular sieves. These “native” species do not enter polyacrylamide gels (7.5%) and are disrupted by sonication. Considering their sedimentation constant, this is only compatible with extremely elongated structures. Another class of acetylcholinesterase species behave like normal globular proteins; we therefore denote them G: purified enzyme (Gp: 11.1 S), tryptic enzyme (Gt: 11.8 S), and another species obtained from A after sonication (G's: 7.7 S). The relationships of A with Gt and G's are discussed, as well as the possible significance of an asymmetric structure for a membrane‐bound enzyme.
Effects of Ring Substituents on the Activity of Phenols as Inhibitors and Uncouplers of Mitochondrial Respiration
Authors: Stockdale Martin , Selwyn Michael J.
Published: March 2005

The effects of substituents on the activity of phenols as uncouplers and inhibitors of mitochondrial respiration have been examined. These activities have been correlated with constants describing the effects of the substituents on the electronic and hydrophobic‐bonding properties of the phenol. The coefficients in these correlation equations have been compared with each other and with the coefficients in similar correlations for the effects of phenols on the solubilised ATPase from mitochondria, and their activity in mediating proton conduction across artificial lipid membranes and as inhibitors of a variety of enzymes.

Differences in the coefficients in the correlation equations for uncoupling and inhibition of respiration indicate that these depend on different properties of the phenol and hence that they are separate effects and that inhibition of substrate accumulation is not a satisfactory explanation for the inhibition of respiration.

High concentrations of uncouplers induce swelling of mitochondria. In the case of 2,4‐dibromophenol, or dicoumarol, this occurs at concentrations similar to those producing inhibition of respiration. With other phenols inhibition of respiration is observed at concentrations lower than those required to induce swelling.

The observations reported here support the hypothesis that phenols uncouple by mediating proton conduction across the inner mitochondrial membrane and that, in general, the inhibition of respiration by high concentrations of phenols is due to direct action on a protein component of the respiratory chain.
Studies on Ferricytochrome c
Authors: Wilson Michael T. , Greenwood Colin
Published: March 2005

A model is proposed to explain the biphasic reduction of ferricytochrome c at alkaline pH assuming an equilibrium distribution between a reducible and non‐reducible conformer. An analysis of this model is given in an appendix to this paper.

Protein denaturants, increasing pH and temperature are found to disturb the conformer equilibrium in favour of the irreducible form. The thermodynamic parameters governing this equilibrium have been determined.

Kinetic parameters defined in the model were measured under a variety of conditions.

The kinetic and equilibrium data strongly suggests that only the reducible conformer possesses the 695 nm band.

Possible origins of the 695 nm band are discussed.
Cycloheximide Resistant Incorporation of Amino Acids into a Polypeptide of the Cytochrome Oxidase ofNeurospora crassa
Authors: Weiss Hanns , Sebald Walter , Bücher Theodor
Published: March 2005

Radioactive leucine was incorporated byNeurospora crassamitochondriain vivoin the presence of cycloheximide. When the membrane protein of these mitochondria was chromatographically separated on oleyl polymethacrylic acid resin, a number of fractions were obtained which differ with respect to their contents of radioactivity and cytochromes. The highest specific radioactivity was found in the fraction containing cytochromeaa3. This fraction proved to be a pure and enzymatically active cytochrome oxidase. Its ratio of absorbance at 280 nm (ox)/443 nm (red.) was 2.1.By means of sodium dodecylsulfate gel‐electrophoresis, this enzyme was separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioactivity.
Deterioration of Rat‐Liver Mitochondria during Centrifugation in a Sucrose Gradient
Authors: Wattiaux Robert , Wattiaux‐De Coninck Simone , Ronveaux‐Dupal Marie‐France
Published: March 2005

The distribution of mitochondrial enzymes after isopyenic centrifugation of a rat‐liver mitochondrial fraction in a sucrose gradient depends on the speed of centrifugation. From the distribution changes, it may be deduced that when centrifugation reaches a certain speed there is an increase in the equilibrium density of some mitochondria; after this the mitochondria undergo disruption. The hydrostatic pressure to which the granules are subjected in the gradient seems to be the main cause of these phenomena. The results of morphological examinations are in good agreement with those suggested by biochemical determinations.
Relation of Toxicity and Conformation of Phallotoxins as Revealed by Optical Rotatory Dispersion Studies
Authors: Faulstich Heinz , Wieland Theodor
Published: March 2005

Natural variants and chemically modified derivatives of phalloidin have been found or prepared which show all degrees of toxicity. High toxicity persists if only three of the side chains are modified. Acetylation of two of the four OH groups of phalloidin leads to less toxic products, the triacetyl derivative is nontoxic. On tosylation toxicity disappeared after reaction of only two OH groups. Alkylation of the indole nitrogen leads to equally or less toxic (N‐methy), ‐ethyl, carbamoylmethyl, ‐tert.butyl) or nontoxic (N‐propyl, ‐pentyl) products. Toxicity is also completely absent if only one of the two rings of the molecule is intact.

In optical rotatory dispersion spectra all of the toxic compounds show in aqueous solution two cotton effects centered around 300 nm and 248 nm, whereas in nearly all of the nontoxic derivatives at least one of the effects is missing. Triacetylphalloidin and tribromophalloidin, both nontoxic, also exhibit “toxic” curves in water. Their conformation, however, changes on going to organic solvents like methanol or acetonitril as observed by optical rotatory dispersion studies. Thus, stability of the “aqueous” conformation in a lipophilic environment is apparently the second prerequisite for a toxic action of phallopeptides.
Mutations habilitant Escherichia colià croître sur acides gras moyens
Authors: Vanderwinkel Edgard , de Vlieghere Monique , Vande Meerssche Jacques
Published: March 2005

New spontaneous mutants of Escherichia coli able to use butyrate or valerate as carbon source have been studied for their growth, adaptation lag and the level of enzymes which may participate in the utilisation of these compounds.

Each mutant acquires the simultaneous constitutivity for uptake of these fatty acids, CoA transferase and high thiolase activities. This mutation designated as butR does not affect the regulation of the β‐oxidation nor that of the glyoxylate cycle.

Induction or mutation for constitutivity (oleR) of β‐oxidation act multiplicatively on the level of thiolase derepressed by the butR mutation.

If the presence of CoA transferase, thiolase and C4–C5 fatty acids entry system is a prerequisite of the growth ability on these fatty acids, the genetic constitutivities (and not the preinduction) of the β‐oxidation (oleR) and particularly of the glyoxylic cycle (aceD) greatly increase the growth capacity. This implies that not only the C, and C, fatty acyl derivatives are very poor inducers of β‐oxidation, as already reported by others, but that some products of their catabolism repress the glyoxylic cycle.

The acquisition of the new growth capacity is thus accomplished by spontaneous mutations affecting regulatory processes; one of these discloses activities which are cryptic in the normal parental strains; the physiological significance of these enzymatic potentialities in wild strains and the mechanism of the interaction between two different regulatory units brought to light here are some of the open issues.
Quantitative Comparison of the Binding of Various Glycolytic Enzymes to F‐Actin and the Interaction of Aldolase with G‐Actin
Authors: Arnold Heidwolf , Henning Roland , Pette Dirk
Published: March 2005

The binding to F‐actin of several crystalline rabbit muscle enzymes of glycogenolysis and glycolysis, was investigated in vitro. Under the conditions chosen, no binding occurs in the case of glycogen phosphorylase, phosphoglucomutase, glycerate phosphomutase and enolase. Aldolase, pyruvate kinase and triosephosphate dehydrogenase are strongly bound to F‐actin, whereas lactate dehydrogenase and phosphoglycerate kinase are bound to a lesser degree. The quantitative differences in the affinity to F‐actin are expressed by differences in the determined binding constants. Analytical evaluation of the binding characteristics suggests two binding sites in the case of aldolase and pyruvate kinase. Analytical ultracentrifugation studies and density gradient centrifugation in sucrose revealed a complex formation between aldolase and G‐actin monomer. An average s20, w value of 9.2 S was obtained for the complex. At higher aldolase concentration, a gelation of the G‐actin was observed which resembles the action of α‐actinin.
A Study of the Reduction and Oxidation of Human Ceruloplasmin
Authors: Carrico Robert J. , Malmstrüm Bo G. , Vänngård Tore
Published: March 2005

The stopped‐flow technique was utilized to measure the 610 and 340 nm absorbances of human ceruloplasmin during oxidation and reduction reactions. The results confirm earlier observations that the chromophores giving rise to these absorbances are different electron acceptors. Under steady state conditions both chromophores were partially reduced which indicates that they participate in the oxidase reaction. Oxidation rates for the 610 and 340 nm chromophores of completely reduced ceruloplasmin were too high to be compatible with the respective steady state absorbances. These results, together with a comparison of the oxidation rates for Type 1 copper of partially and completely reduced protein, showed that the oxidation rate for one chromophore is influenced by the oxidation states of the other and/or the nonchromophoric paramagnetic copper (Type 2). Therefore, the oxidase mechanism appears to involve concerted transfer of electrons from the 610 and 340 nm chromophores and possibly Type 2 copper.
Isolation and Properties of tRNA Nucleotidyl Transferase from Yeast
Authors: Sternbach Hans , von der Haar Friedrich , Schlimme Eckhard , Gaertner Erika , Cramer Friedrich
Published: March 2005

tRNA nucleotidyl transferase has been purified 5000‐fold from commercial bakers' yeast. The isolation procedure includes ammonium sulfate fractionation and chromatography on DEAE‐cellulose, CM‐cellulose, Biogel and phosphocellulose.

The enzyme is homogeneous on analytical centrifugation and by sodium dodecylsulfate gel electrophoresis.

The molecular weight of the native and denatured enzyme Mc=Or is 71000 as determined by ultracentrifuge measurements and 70000 as determined by sodium dodecylsulfate gel electrophoresis.

On micro isoelectric focusing the enzyme shows an isoelectric point at about pH 7.5.

The enzyme catalyses the incorporation of ATP and CTP into the 3′‐end of tRNA, and, in the presence of pyrophosphate, the pyrophosphorolyses of adenylic and cytidylic acid residues from the 3′‐end of tRNA.
Mise en évidence et analyse des ribosomes et des RNA associés aux mitochondries de cellules de mammifères
Authors: Eladari Marthe‐Elisabeth , Galibert Francis , Boiron Michel
Published: March 2005

Mitochondrial fraction prepared from mammalian cells (rat liver) by high and slow speed centrifugation and purified by sedimentation in an isopycnic sucrose gradient are shown to contain ribosomes.

These ribosomes have a sedimentation coefficient of 83 S (measured in the absence of KCl) and a buoyant density of 1.54 g/cm3 as compared to 80S and 1.55 g/cm3 for cytoplasmic ribosomes.

The sedimentation behaviour of mitochondrial ribosomes varies as a function of KCl concentration. The dissociation of cytoplasmic and mitochondrial ribosomes into sub‐units begins towards 0.1 M KCl and is complete in 1 M KCl. The sedimentation coefficient of the intact ribosomes and their dissociated units fall as the concentration of KCl is increased between 0 and 1 M.

RNA prepared from rat liver mitochondrial fraction contains a low molecular weight component and two high molecular weight species whose sedimentation coefficients 26 S and 17 S correspond to molecular masses of 1.51 × 106 and 0.61 × 106 daltons. These mitochondrial RNA can be separated by sucrose gradient sedimentation from cytoplasmic ribosomal RNA species sedimenting at 28 S and 18 S.

Mitochondrial RNA were also characterized by acrylamide gel electrophoresis. In 2.50% gels, the electrophoretic mobility of high molecular weight mitochondrial RNA was found to be slightly higher than that of their cytoplasmic counterparts. Moreover, the RNA corresponding to the smaller units was separated into two distinct peaks.

Electrophoresis in 8% acrylamide gels revealved 5 S and 4 S RNA with a migration pattern similar to that of cytoplasmic 5 S and transfer RNA but present in different amounts and additional RNA with an electrophoretic mobility of 0.31 and 0.85 if that of 4 S RNA is equalled to 1.
Fatty Acid Synthetase from Pig Liver
Authors: Dutler Hans , Coon Minor J. , Kull Arthur , Vogle Hugo , Waldvogel Guy , Prelog Vlado
Published: March 2005

An enzyme, exhibiting NADPH‐dependent oxidoreductase activity towards alicyclic ketones, has been extracted from pig liver and purified 122‐fold with respect to the protein contained in the crude extract after centrifugation at 54000×g. General properties, ultraviolet spectrum, stability, kinetic constants (V and Km) for NADPH and for typical substrates are reported. The molecular weight of the enzyme was estimated at 500000 by gel‐filtration. The enzyme is Hs(HB)‐specific with respect to coenzyme. Alicyclic ketones can be conveniently used to measure the activity at all stages of purification. The topography of the active site responsible for oxidoreductase activity has been investigated by use of rigid alicyclic ketones such as trans‐decal‐1‐ones as probes. In the accompanying paper it is shown (1) that the biological function of the whole enzyme complex is that of a fatty acid synthetase and (2) that the oxidoreductase activity can be ascribed to its 3‐oxoacyl‐acyl‐carrier protein reductase component.
Multiple Carboxymethylation of Histidines in Bovine Ribonuclease A
Authors: Bello Jake , Nowoswiat Eugène F.
Published: March 2005

Reaction of RNAase A with bromoacetate at pH 5.5 for 1–42 days results in multiple reactions. Alkylation of residues proceeds in the sequence: (1) N‐1 of histidine‐119; (2) methionine (probably methionine‐30); (3) N‐3 of histidine‐12; (4) N‐3 of histidine‐105 and N‐3 of 1‐carboxy‐methyl histidine‐119; and (5) lysine‐1. Both histidine‐12 and histidine‐119 of the same active site are carboxymethylated. A derivative carboxymethylated at both active site histidines is obtained in 1 day and probably some of this derivative is obtained in short reaction times. This is contrary to the conclusions of earlier investigations. Histidine‐48 undergoes little or no reaction. The results are in accord with the X‐ray structure of RNAase.
Studies on the Biosynthesis of NAD from Nicotinamide and on the Intracellular Pyridine Nucleotide Cycle in Isolated Perfused Rat Liver
Authors: Keller Jürgen , Liersch Michael , Grunicke Hans
Published: March 2005

Isolated perfused rat liver incorporates nicotinamide at a concentration of 10 μM into NADvianicotinamide mononucleotide. A deamidation of nicotinamide could not be demonstrated.

Evidence is presented which indicates that the exchange reaction catalyzed by NAD glycohydrolase is not involved in the uptake of nicotinamide into NAD.

At the concentrations used, the rate of the incorporation of nicotinamide into NAD is about 40% of the incorporation rate observed with nicotinic acid.

Results obtained after perfusion with [14C]nicotinic acid support the concept of an intracellular pyridine nucleotide cycle. However, in contrast to the general opinion, our results demonstrate that in rat liver nicotinamide generated by NAD breakdown is not deamidated but reutilizedvianicotinamide mononucleotide.

After perfusion with 0.1 mM [14C]nicotinamide for 2.5 h, less than 2% of the total radioactivity is excreted into the bile. The same value is obtained after perfusion with 50 μM [14C]nicotinic acid. From these results it is concluded that the excretion of nicotinamide by the liver and the subsequent deamidation within the intestinal tract which have been considered important in the utilization of nicotinamide by the liver, are without biological relevance at normal nicotinamide concentrations.
Synthetic Polynucleotides
Authors: Kössel Hans , Roychoudhury Ranajit
Published: March 2005

The enzyme terminal deoxynucleotidyl transferase catalyzes the addition of one or two riboadenylic acid residues to deoxyoligonucleotides. This reaction allows the specific labelling of deoxyoligonucleotides at the 3′‐ends if [α‐32P]ATP is used as substrate.

In order to eliminate the product of the addition of two riboadenylic acid residues the total reaction mixtures after incubation with terminal transferase are treated with alkali and subsequently with phosphatase. Deoxyoligonucleotidyl [32P]pAr is then obtained as the sole reaction product. Digestion with spleen phosphodiesterase yields the deoxynucleotide‐3′‐[32P]monophosphates corresponding to the 3′‐ends of the deoxyoligonucleotides.

The riboadenosyl residues at the 3′‐termini of the monoaddition products also can be eliminated by treatment with periodate and cyclohexylamine whereupon deoxyoligonucleotides specifically labelled at the 3′‐termini with [32P] phosphomonoester groups are obtained.

The sensitivity of the method and the faithfulness in the identification od the 3′‐terminal deoxynucleoside residues could be tested using as little as 0.1 A260 units (5 μg) of oligodeoxynucleotides of specific base sequences.
Isolation and Characterization of Keto‐Carotenoids from the Neutral Extract of Algal Mat Communities of a Desert Soil
Authors: Bauman Albert J. , Boettger Heinz G. , Kelly Ann M. , Cameron Roy E. , Yokoyama Henry
Published: March 2005

The carotenoid pigments of surficial algal mat communities of a California absolute subtropical desert were isolated and characterized principally by means of high resolution mass spectrometry. The pigments were all oxidized keto‐types, predominantly canthaxanthin, its isomers, and echinone. The carotenoid pattern suggests that the mats represent an old climax ecosystem in which the algal cells at the time of collection were starved for nitrogen.
The Differentiation of Stereoheterotopic Groups
Authors: Hirschmann Hans , Hanson Kenneth R.
Published: March 2005

Constitutionally equivalent groups that occur in a single molecule are classified on the basis of their topic relationships by comparing their environments. The proposed classification extends earlier suggestions of Mislow and Raban. The existence of homotopic groups (those indistinguishable under any conditions) is related to the symmetry properties (gyrosymmetry) of rigid as well as flexible finite molecules. Stereoheterotopic groups (those that are distinguishable but occur in constitutionally equivalent environments) may be associated with various steric elements which will be fully discussed in a second paper. Each of these elements, the center, the axis, the plane and the torsional element, is divided into four mutually exclusive classes: the chiral and achiral elements of stereoisomerism; the prochiral and proachiral elements of prostereoisomerism. Of these classes, only the various centers of stereoisomerism and of prostereoisomerism are discussed in this report. Some modifications in our earlier proposals for the naming of individual stereoheterotopic groups are introduced. The utility of these new and revised terminologies for biochemical discussions is indicated.
Structural Investigations on the Core Polysaccharide of Escherichia coli 0100
Authors: Hämmerling Günter , Lüderitz Otto , Westphal Otto , Mäkelä P. Helena
Published: March 2005

In order to investigate the core structure of the lipopolysaccharide from an Escherichia coli 0100 strain, two derivatives were prepared from this strain by introducing genetic material from Salmonella abony. One, an SR hybrid, contained a lipopolysaccharide with short side chains consisting of simple repeating units of Salmonella group B specificity attached to the E. coli core. The other, an R form, contained only the E. coli core and no O side chains, due to a mutation in the Salmonella rfb chromosomal region, which determines the synthesis of the O repeating units.

By partial acid hydrolysis of the SR and R lipopolysaccharides, oligosaccharide fragments were obtained which made it possible to reconstruct the hexose region of the core (see formula below). Methylation studies performed on the dephosphorylated polysaccharides confirmed and extended these results. It was found that only about 60% of the distal glucose units of the core chains were substituted by N‐acetylglucosamine residues. Further, the heptose region was shown to form a branched trisaccharide.

On mild acid hydrolysis of both lipopolysaccharide preparations a Gal‐α1,7(8)‐KDO disaccharide was formed, indicating that a galactose unit is linked to the 2‐keto‐3‐deoxyoctonate (KDO) portion of the lipopolysaccharide.

After Smith degradation of the SR lipopolysaccharide, the oligosaccharide Gal‐β1,4‐Glc‐glycerol was identified which derived from the linkage region between the Salmonella type repeating unit and the E. coli core. Only the core stubs substituted by N‐acetylglucosamine residues carried an O‐specific unit.

On the basis of the these findings the partial structure of the E. coli 0100 core polysaccharide with a Salmonella group B‐specific unit attached to it is proposed as given below. The relationships to core structures identified in lipopolysaccharides of other representatives of Enterobacteriaceae are discussed.
A Calorimetric Study of the Denaturation of Lysozyme by Guanidine Hydrochloride and Hydrochloric Acid
Authors: Lapanje Savo , Wadsö Ingemar
Published: March 2005

The denaturation of lysozyme by guanidine hydrochloride and hydrochloric acid at several temperatures, and by guanidine hydrochloride alone at 25°C was studied using a microcalorimetric technique. From results of the calorimetric measurements at 25, 29.5 and 34.6°C enthalpies of denaturation by hydrochloric acid at constant guanidine hydrochloride concentration were calculated. From these ΔH values corresponding ΔCp values were calculated.

An approximate value for the enthalpy of denaturation at 25°C of lysozyme by guanidine hydrochloride alone has also been obtained. It is shown that microcalorimetry is a sensitive method for following the two denaturation processes.
Purification and Properties of Rat‐Liver‐Mitochondrial Adenosine Triphosphatase
Authors: Lambeth David O. , Lardy Henry A.
Published: March 2005

Mitochondrial ATPase from rat liver has been highly purified to a maximum specific activity of 110 in Tris‐bicarbonate buffer at pH 8.0. Sedimentation velocity studies in the analytical ultracentrifuge show that the enzyme sediments as a single symmetrical peak with a sedimentation coefficient (so20, w), of 12.9 S. Molecular weight determinations by both high‐speed sedimentation equilibrium and polyacrylamide gel chromatography indicate a molecular weight of 360000 ± 10000. Polyacrylamide gel electrophoresis of the enzyme dissociated by sodium dodecyl sulfate reveals one major and three minor bands with molecular weights of 53000, 28000, 12500, and 8000–9000. A calculated molecular weight of approximately 365000 is obtained by assuming the presence of six subunits of molecular weight 53000 and one of each of the lighter subunits. Like the beef‐heart enzyme, rat‐liver ATPase activity is lost on incubation at 5°C and examination by high‐speed sedimentation equilibrium at 5°C indicates that the molecule is extensively dissociated to components of low molecular weight. The enzymatic activities of both rat‐liver‐mitochondrial and beef‐heart‐mitochondrial ATPases are sensitive to the anion of the buffer used at pH 8.0 with activity decreasing in the following series: HCO3,− > maleate > chloride > acetate = sulfate. Sulfite, chromate and dinitrophenol stimulate the activity of either enzyme in Tris‐sulfate buffer, but only sulfite increases the activity seen in Tris‐bicarbonate buffer. Aurovertin decreases the enzymatic activity to approximately the same level, regardless of the anion(s) present. The enzyme is insensitive to oligomycin.
Proteolytic Cleavage of Native DNA Polymerase into Two Different Catalytic Fragments
Authors: Klenow Hans , Overgaard‐Hansen Kay , Patkar Shamkant A.
Published: March 2005

Treatment of native DNA polymerase from Escherichia coli with subtilisin may lead to its cleavage into a polymerase fragment and an exonuclease fragment. The cleavage has been followed by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulfate and by the change both in polymerase activity and in exonuclease activity. The electrophoretic analysis has shown that subtilisin treatment leads to the formation of two new protein bands and simultaneous disappearance of the band corresponding to the native enzyme. The cleavage of the native polymerase may be accompanied by change in the polymerase activity. The change appears to depend on the conditions of the polymerase assay. With activated calf thymus DNA the activity increases by a factor of up to 2.3 when assayed at low phosphate buffer concentration. At higher buffer concentration in the assay mixture the cleavage gives rise to a decrease in polymerase activity. With poly[d(A‐T)] · poly[d(A‐T) · the cleavage gives rise to a decrease in polymerase activity at both low and high buffer concentration. The effect of buffer concentration on the polymerase activity of the native and the cleaved enzyme showed that maximum activity for the two enzymes was obtained at different buffer concentrations in the presence of activated calf thymus DNA. In the presence of poly[d(A‐T)] · poly[d(A‐T)] maximum activity was obtained at the same buffer concentration but the activity of the cleaved enzyme was lower than that of the native at all buffer concentrations. A similar differential effect of ionic strength on the activity has been found for the exonuclease site of the native enzyme and the cleaved exonuclease fragment. This differential effect depends, however, on the molecular size of the substrate poly[d(A‐T)] · poly[d(A‐T)]. It appears, furthermore, that the exonuclease site of the cleaved exonuclease fragment has a smaller affinity towards poly[d(A‐T)] · poly[d(A‐T)] than that of the native enzyme. Finally, while the exonuclease activity of the native enzyme towards poly[d(A‐T)] · poly[d(A‐T)] is stimulated by conditions that permit simultaneous synthesis no such synthesis stimulated exonuclease activity is observed after cleavage of the native enzyme into a polymerase fragment and an exonuclease fragment.
Quantitative Studies on the Synthesis in vivo of Prostaglandins in the Rat
Authors: Gréen Krister , Samuelsson Bengt
Published: March 2005

The excretion of two metabolites of prostaglandin E2 (tetranor‐prostaglandin B1 and tetranor‐prostaglandin E1) in rat urine has been determined using an electron capture technique. The daily excretion in female rats ranged from 2.16 to 3.93 μg/kg and in male rats from 2.04 to 2.63 μg/kg. When the rats were exposed to cold stress excretion of the metabolites increased 2 to 2.5‐fold.
A Study of Mitochondrial Protein Synthesis in Intact HeLa Cells
Authors: Brega Agnese , Baglioni Corrado
Published: March 2005

Mitochondrial protein synthesis has been studied in intact HeLa cells by utilizing specific inhibitors. We have observed that in the presence of pederine, an inhibitor of cytoplasmic protein synthesis in mammalian cells, protein synthesis continues for at least 1 h at approximately 0.5% of the rate of untreated cells. When control cells and cells treated with pederine are fractionated after an incubation with labeled amino acids into a mitochondrial and a supernatant fraction, it is found that incorporation into the mitochondrial fraction is higher in the presence of the inhibitor. Mitochondrial protein synthesis has been shown to take place on aggregates of mitochondrial ribosomes.

The synthesis of mitochondrial proteins has been studied by incubating HeLa cells with labeled amino acids and either pederine or chloramphenicol, an inhibitor of mitochondrial protein synthesis. We have been able to establish that the structural protein of mitochondrial ribosomes is synthesized on cytoplasmic ribosomes and its synthesis is thus inhibited by pederine, but not by chloramphenicol. Mitochondrial proteins have been analyzed by acrylamide gel electrophoresis after fractionation of the mitochondria into inner and outer membranes. Proteins synthesized by mitochondria in the presence of pederine are located predominantly on the inner mitochondrial membranes. Only few protein species synthesized by mitochondria are resolved by gel electrophoresis. This finding is discussed in relationship to the information content of mitochondrial DNA.
Photoreactions of Chloroplasts in a Glycine Medium
Authors: Walz Dieter , Schuldiner Shimon , Avron Mordhay
Published: March 2005

The rate of photoinduced electron transport in a medium containing glycine as the major salt was very low. It was dramatically increased by low concentrations of monovalent salts (2–4 mM) to rates typical of uncoupled systems. Further increase in salt concentration (beyond 15–20 mM) cause a sharp decrease back to “coupled” values. Salts of divalent ions, like Mg2+ were even more effective in the “recoupling” process (0.1 mM).

When low amounts of Mg2+, phosphate and ADP were added to the glycine medium, photophosphorylation proceeded initially at high rates but stopped after a certain amount of ATP was accumulated. This effect was traced to a complete inhibition by the ATP formed of any further phosphorylation. The concentration of ATP needed to inhibit increased sharply with an increase of the salt concentration in the medium. ADP exhibited a similar inhibitory effect but higher concentrations were required.

At concentrations where ATP inhibited phosphorylation, proton uptake was not affected.
Effects of Iodination and Acetylation of Tyrosyl Residues on the Activity and Structure of Arginine Kinase from Lobster Muscle
Authors: Fattoum Abdellatif , Kassab Ridha , Pradel Louise‐Anne
Published: March 2005

The iodination and O‐acetylation of tyrosyl residues in arginine kinase from lobster muscle (Homarus vulgaris) with iodine and N‐acetylimidazole have been studied after reversible blocking of the thiol groups.

The conversion of one tyrosyl residue to monoiodotyrosine, as measured by spectrotitration and by radioactive iodine labeling, resulted in a total loss of activity.

The extent of the conformational changes occurring after nitration by tetranitromethane and iodination was studied by the optical rotary dispersion and the hydrogen‐tritium exchange techniques.

The functional properties of this tyrosyl residue were underlined by the inhibition of arginine kinase after o‐acetylation and the ability of the modified protein to recover full activity upon tyrosyl de‐o‐acetylation with neutral hydroxylamine.
Studies on Lichen Enzymes
Authors: Mosbach Klaus , Schultz Joachim
Published: March 2005

Orsellinate decarboxylase, catalyzing the decarboxylation of orsellinic acid (2,4‐dihydroxy‐6‐methyl benzoic acid) to orcinol has been isolated from the lichen Lasallia ptulata.

The substrate specificity of the enzyme has been tested: only orsellinic acid, 3‐chloro‐, 5‐chloro‐orsellinic acids, homo‐orsellinic acid (2,4‐dihydroxy‐6‐ethyl benzoic acid) and everninic acid (4‐methoxy‐2‐hydroxy‐6‐methyl benzoic acid) were decarboxylated.

The obtained degree of purification was 400‐fold with a hal specific activity of 14.1 units/mg of protein. The purified enzyme has a molecular weight of about 72000 (±10%) as determined by dodecyl sulphate‐polyacrylamide gel electrophoresis. The Kmfor orsellinic acid has been determined as 211 μM, the Ki of the inhibitor β‐resorcylic acid (2,4‐dihydroxy benzoic acid) as 800 μM. A spectrophotometric method suitable for the assay of orsellinate decarboxylase is described.
Fingerprint Maps of Tryptic Peptides from Subunits of Escherichia coli and T4‐Modified DNA‐Dependent RNA Polymerases
Authors: Schachner Melitta , Zillig Wolfram
Published: March 2005

DNA‐dependent RNA polymerase from bacteriophage T4‐infected Escherichia coli cells differs in all its subunits from the host's core enzyme.

T4‐modified α differs from E. coliα by 1 or 2 additional peptides. One additional tyrosine positive peptide and the displacement of one histidine containing peptide is characteristic for T4‐modified β'. T4‐modified β is significantly different from E. coliβ in its histidine and tryptophan containing peptides, whereas differences in the tyrosine peptide pattern are less obvious.

No common features were observed among the peptide patterns of the subunits from E. coli RNA polymerase.
Regulation of the Cytoplasmic Inorganic Pyrophosphatase of Rhodospirillum rubrum
Authors: Klemme J.‐H. , Gest H.
Published: March 2005

This study is concerned with the catalytic and regulatory properties of soluble inorganic pyrophosphatase of the nonsulfur purple photosynthetic bacterium Rhodospirillum rubrum. A procedure is described for reproducible 100‐fold purification of the enzyme, which shows complex kinetic behavior. Free Mg2+ is an activator of the pyrophosphatase, and virtually eliminates the sigmoidicity otherwise observed in the substrate (magnesium pyrophosphate) saturation kinetic curves. In the absence of appreciable free Mg2+, the enzyme is inhibited by 2‐phosphoglyceric acid, which has the effect of increasing the [S]0.5 value (substrate concentration required for half‐maximal velocity) and of decreasing V. The pyrophosphatase, in dilute solution, is slowly inactivated when the pH is less than 8.6, and this is specifically prevented by Zn2+. Inactivation caused by exposure to low pH (7.6–8.2) can be reversed partially by addition of Zn2+ and/or exposure to high pH (9–9.5). Full reactivation can be achieved by proper adjustment of pH and Zn2+ concentration, together with addition of a reductant (dithionite). The pH‐induced inactivation is accelerated by nucleoside triphosphates, reduced pyridine nucleotides, and the allosteric inhibitor, 2‐phosphoglyceric acid. Such inactivations are reversible, and are not accompanied by change in molecular weight of the enzyme (∼100000). It appears that the R. rubrum inorganic pyrophosphatase can exist in several interconvertible states, which are characterized by inherently different specific activities. Transitions between alternative states are influenced by the various chemical modifiers and conditions noted, and the phenomena described provide a basis for further investigation of the mechanisms responsible for regulation of the enzyme's activity.
Coenzyme Binding in Yeast Pyruvate Decarboxylase
Authors: Wittorf John H. , Gubler Clark J.
Published: March 2005

A kinetic investigation of the behavior of apopyruvate decarboxylase from Saccharomyces carlsbergensiswith thiamine pyrophosphate analogues gave the values of 23 μM as the Km for thiamine pyrophosphate and 20 μM as the Km for 2′‐ethylthiamine pyrophosphate. The Vfor the latter compound was 25%, that of thiamine pyrophosphate. Inhibitor constants, Ki, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme, (the values are for the pyrophosphate esters): tetrahydrothiamine, 6.5 μM; oxythiamine, 20 μM; 2′‐n‐butyIthiamine, 45 μM; 2′‐methoxythiamine, 70 μM; pyrithiamine, 78 μM; 2′‐demethylthiamine, 220 μM; 2′‐hydroxythiamine, 380 μM. None of the inhibitors exhibited coenzyme activity.

A hydrophobic interaction of the 2′‐methyl group of thiamine pyrophosphate with the apoenzyme has been proposed and a model for the coenzyme binding site of pyruvate decarboxylase, consisting of a pyrophosphate and a pyrimidine binding region, is discussed.
An Improved Technique for the Analysis of Ribonucleoprotein Fragments from Escherichia coli 30S Ribosomes
Authors: Brimacombe Richard , Morgan Joan M. , Cox Robert A.
Published: March 2005

30S ribosomal subunits from Escherichia coli were hydrolyzed with ribonuclease T1, and the ribonucleoprotein fragments produced were fractionated on a 5% polyacrylamide/0.5% agarose composite gel. Two main breakdown products were observed corresponding in size to about 20% and 60% of the intact 30S particle.

Each fraction from the 5% composite gel was subjected to protein analysis on a periodatesoluble 15% polyacrylamide gel, run in the detergent Sarkosyl. The smaller ribonucleotprotein fragment was found to contain only four of the 30S ribosomal proteins, and these were present in equimolar amounts. The larger fragment had a complex protein composition; it was deficient in the four proteins contained in the smaller fragment, and was considerably enriched in the largest 30S protein.

The results show a striking correlation with Nomura's “assembly map” of the 30S particle.
Inactivation of 50S Ribosomal Subunits by 2‐Methoxy‐5‐nitrotropone
Authors: Suzuka Iwao
Published: March 2005

Treatment of 50S ribosomal subunits with 2‐methoxy‐5‐nitrotropone results in extensive loss of their ability to synthesize polyphenylalanine, display ribosome‐dependent GTPase activity, or bind GTP to ribosomes. In contrast, 5‐nitrotroponyl 50S subunits are fully active for diphenylalanine synthesis. Thus, 2‐methoxy‐5 nitrotropone‐reactable ribosomal protein groups on the 50S subunit may be associated with translocation activity of ribosomes.

Sedimentation analysis of 5‐nitrotroponyl 50S subunits at 0.02 M Mg2+ shows a slightly faster sedimenting component (54S) and their dimerized form (79S). Furthermore, the particles which are reassociated with 30S and 5‐nitrotroponyl 50S subunits are dissociated into each subunit under conditions of Mg2+ concentration (5 mM) where most of 30S‐associated control 50S particles are still present as a 70S particle. These results appear to reflect the structural alteration of the 50S subunits caused by the treatment of 2‐methoxy‐5‐nitrotropone.
A Quantitative Analysis of Proflavine Binding to Polyadenylic Acid, Polyuridylic Acid, and Transfer RNA
Authors: Dourlent Maurice , Hélène Claude
Published: March 2005

Absorbance measurements show that the binding of proflavine to poly (A) and poly(U) can be quantitatively resolved (within ± 10% relative error) by the consideration of two bound species whose spectral properties and mechanisms of interaction are similar to those found in the DNA. proflavin systems: cooperative binding along the phosphate backbone (process I) and interaction between the aromatic rings of base and dye resulting presumably from some kind of interacalation of the ligand between consecutive bases (process II). The comparison with a simple theoretical model shows that process I and process II are not independent but coupled.

The spectrophotometric method is able to resolve the binding of proflavine to a tRNA species with the same accuracy as that obtained for poly(A) and poly(U). Accordingly, it is proposed that the mode of interaction between proflavine and all classes of polynucleotides (DNA, RNA, synthetic polymers) is similar in nature. From the differences observed in the distribution of the bound dye between the two complexes, the existence of a close correlation between the secondary structure of the polymer and its ability for binding the dye in complex II is proposed.
Fate of Exogenous DNA in Arabidopsis thaliana
Authors: Ledoux Lucien , Huart Raoul , JACOBS Michel
Published: March 2005

It has been shown that when Arabidopsis seeds are incubated with different heteropycnic labelled DNA during the first 4 days of germinating, large fragments of exogenous DNA are taken up by the seedling plant tissues (cotyledons and rootlet) and remain polymerized and double‐stranded during the entire growth period. Only a small percentage of the population of exogenous DNA molecules appears to be destroyed and reutilized for DNA synthesis de novo. Most of the exogenous DNA remains free or becomes integrated with the Arabidopsis DNA as double‐stranded molecules, not separable by alkali denaturation, but easily separable by ultrasonication.

Analysis of different tissues and organs of growing plants reveals that a large amount of foreign DNA remains in the cotyledons during the vegetative growth phase, but is redistributed at the flowering stage (when cotyledons etiolate). At this time, the foreign DNA leaves the cotyledons and migrates towards the flowering buds where it accumulates as a highly molecular and double‐stranded material. In the F1 progeny, radioactive molecules are found which do not correspond to the endogenous material and which have a density depending on that of the foreign DNA used.