bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Distinctive Molecular Forms of Thymidine Kinase in Mitochondria of Normal and Bromodeoxyuridine‐Resistant HeLa Cells
Authors: Kit Saul , Leung Wai‐Choi , Kaplan Lawrence A.
Published: March 2005

Disc polyacrylamide‐gel electrophoresis analyses have been carried out on the dT kinase activities of “cytosol” (high‐speed supernatant) and mitochondrial fractions of parental HeLa S3 and enzyme‐deficient HeLa(BU25) cells. The HeLa S3 cytosol fraction contained a very active dT kinase molecular form with a mobility relative to the tracking dye (Rx) of about 0.25. Conversion of HeLa S3 cells to dT‐kinase‐deficient HeLa(BU25) resulted in the loss of most of the cytosol dT kinase activity. The mitochondrial fraction of HeLa(BU25) cells did not contain the 0.25 Rx activity, but did shown 2 more rapidly migrating molecular forms (Rx of 0.4 and 0.6–0.7). The mitochondrial fraction of parental HeLa S3 cells also exhibited the 0.4 and 0.6 to 0.7 Rx dT kinase activities, and addition, contained the 0.25 Rx cytosol‐like enzyme. The distinctive dT kinase molecular forms observed in the mitochondrial fraction account for most of the residual enzyme activity of mutant HeLa(BU25) cells.
Transcription of High‐Molecular‐Weight RNA from Hen‐Oviduct Chromatin by Bacterial and Endogenous Form‐B RNA Polymerases
Authors: Cox Ronald F.
Published: March 2005

With the aid of the ribonuclease inhibitor, heparin, techniques were developed to isolate high‐molecular‐weight RNA products synthesised by Micrococcus and homologous form B RNA polymerases using hen oviduct chromatin as template. These methods are suitable for analysis of RNA transcripts synthesised in vitro by a variety of enzymes and templates.

A highly sensitive assay for ribonuclease, based on the ability of the latter to degrade ovalbumin mRNA, confirmed that chromatin and enzyme samples contained significant amounts of ribonuclease activity.

Heparin completely inhibits initiation of both bacterial and homologous enzymes, but has no effect on elongation. The average rates of elongation of RNA polymerases on chromatin DNA could therefore be determined. These values were 3–6 nucleotides per second for bacterial enzyme and 1–2 nucleotides per second for homologous enzyme.

By allowing initiation of bacterial enzyme prior to the addition of heparin, RNA chains approaching 2000 nucleotides in length (molecular weight 0.7 X 106) were synthesised after 1 min; at later times, these chains exceeded 5000 nucleotides in length.

Hen oviduct chromatin contains endogenous RNA polymerase activity which represents predominantly the elongation of form B enzymes. RNA chains ranging from 100 to 2000 nucleotides long are still attached to these enzymes in isolated chromatin. By allowing elongation of these chains in vitro, products ranging from 2000 to 10000 nucleotides in length (molecular weight 0.7 to 3.0 X 106) were synthesised.

Estimates of the number of endogenous RNA polymerases bound to chromatin DNA suggest that approximately 10% of the total form B RNA polymerase molecules per cell are retained in the isolated chromatin fraction.
Unsaturated Fatty Acids as Reactivators of the Calcium‐Dependent ATPase of Delipidated Sarcoplasmic Membranes
Authors: The Rudy , Hasselbach Wilhelm
Published: March 2005

The reconstituting effect of unsaturated fatty acids whose chain length varied between C6–C24 on delipidated sarcoplasmic membranes has been analyzed.

The reconstituted sarcoplasmic ATPase requires 70–106 mol fatty acid per 100000 dalton for optimal activity.

Half‐maximal activity of the reconstituted sarcoplasmic ATPase is reached between 0.2 μM and 0.6 μM ionized calcium.

The ATPase activities of the reconstituted vesicular preparations show an identical steep decline at pH values > 7, whereas the native preparation still exhibits a considerable activity at pH 8.0.

The most effective reactivating unsaturated fatty acids are those with a chain length of C16–C18 and whose double bonds are in the middle of the chain.
Effect of Temperature on the Behavior of Rat‐Liver Mitochondria during Centrifugation in a Sucrose Gradient
Authors: Wattiaux‐De Coninck Simone , Ronveaux‐Dupal Marie‐France , Dubois Franz , Wattiaux Robert
Published: March 2005

We have investigated the effect of the temperature of centrifugation on the behavior of ratliver mitochondria during isopycnic centrifugation in a sucrose gradient. From the distribution curves of the reference enzymes, it may be deduced that the damaging effect of high‐speed centrifugation on mitochondria can be prevented by increasing the temperature of centrifugation. The results of the morphological examinations are in good agreement with the biochemical determinations. As previously described Wattiaux et al. (1971) the hydrostatic pressure generated by centrifugation is the main causal agent of the deterioration of mitochondria. The effect of temperature suggests that the pressure acts either on the physical state of the mitochondrial membrane lipids or by disrupting hydrophobic bonds essential for the mitochondrial membrane stability. From a practical point of view, our results show that it is possible to prevent the damaging effects of centrifugation on rat‐liver mitochondria by adjusting the temperature.
Characterization of Cd, Zn‐Thionein (Metallothionein) Isolated from Rat and Chicken Liver
Authors: Weser Ulrich , Rupp Heinz , Donay Falk , Linnemann Frithjof , Voelter Wolfgang , Voetsch Walter , Jung Günther
Published: March 2005

The preparation of pure metallothionein from Cd‐pretreated rats and chicken was performed using short‐time treatment with chloroform/ethanol followed by ion‐exchange and gel chromatography. The protein contained 7 g‐atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn‐thionein remained homogeneous during Sephadex G‐25, G‐50 and G‐75 gel filtration, during polyacrylamide disc‐gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one‐third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn‐thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X‐ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ250= 80.6 mM−1· cm−1. Virtually no aromatic amino acids were present. The ɛ250 value of the apoprotein prepared by metal displacement using HCl was ɛ250= 13.2 mM−1· cm−1. When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ250= 18.7 m M−1· cm−1). The existence of inter‐ or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di‐tert‐butyloxy‐carbonyl‐l‐cystine and tert‐butyloxycarbonyl‐l‐cysteine as model compounds for high‐molecular‐weight and water‐soluble cysteine compounds, respectively.

From CD data it was concluded that Cd, Zn‐thionein exists mainly as a random‐coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X‐ray photo‐electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d3/2= 411.0 eV and Cd 3d5/2= 404.4 eV), Zn (2 p3/2= 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn2+ and Cd2+ were 163.0 eV and 162.8 eV, respectively.
Glutamate déshydrogénase
Authors: Dessen Philippe , Pantaloni Dominique
Published: March 2005

The object of this paper is to analyse the effects of the coenzymes NAD(P)+ and NAD(P)H, and the effectors ADP and GTP, on the polyhexameric structure of pig‐liver dehydrogenase. The linear polymerisation model proposed by Eisenberg for the native quaternary structure of this protein is valid with any effector; the observed variations of the degree of polymerization are explained by the modification of the apparent association constant of the hexamers. The appendices I and II define the free and associated areas and give, the theoretical foundations of the variation of the association constant of hexamers in terms of the binding of the ligands to the protomers.

The increase in the degree of polymerization of the glutamate dehydrogenase with the binding of NAD(P)H is explained by a higher affinity of the coenzymes for the protomers which have an associated are compared to the protomers which have a free area. No variation is observed with NAD(P)+, ADP, or GTP alone. The formation of the protein · GTP · NAD(P)H ternary complex leads to a complete depolymerization when the two ligands are in saturating concentrations.

The systematic study of the variations of polymerization in terms of increasing concentration of NAD(P)H at constant concentration of GTP, or in terms of increasing concentration of GTP at constant concentration of NAD(P)H shows that the interaction between the two opposite protomers of two consecutive hexamers is responsible for the sigmoidal shape of the depolymerization curves. The reversibility of this effect by ADP is assigned to a competition between the binding of ADP and the binding of GTP.
Immunochemistry of Tetanus Toxin
Authors: Bizzini Bernard , Turpin André , Raynaud Marcel
Published: March 2005

The tyrosyl residues of tetanus toxin can be specifically nitrated by tetranitromethane. The introduction of NO2 groups into the toxin molecule is followed by a rapid decrease in toxicity. Although tryptophyl residues are differently involved and some aggregation occurs, these two phenomena do not take any significant part in the toxicity loss. The immunological reactivity in vitro is preserved. However, it is modified when a certain amount of NO2 groups have been introduced. The thus‐formed derivative no longer gives a reaction of identity with the native toxin. Furthermore, when a sufficiently large number of NO2 groups have been introduced, two lines of precipitation are seen. Antisera raised in rabbits with a few nitro‐derivatives of the toxin exhibit a low avidity. Precipitating power with pure toxin of these antisera is lower than with a normal antitetanus serum. The capability of tetanus toxin to give rise to neutralizing antibodies is not impaired until 33 NO2 groups have been introduced, then it gradually decreases until it finally disappears. Modification of the toxin by nitration allowed us to discriminate three groups of determinants. In particular, the results presented are further substantiated by those given by 3‐aminotyrosyl derivatives of tetanus toxin.
A Non‐Intercalating Proflavine Derivative
Authors: Müller Werner , Crothers Donald M. , Waring Michael J.
Published: March 2005

A proflavine derivative with bulky substituents, 2,7‐di‐t‐butyl‐proflavine, binds to DNA but apparently not by intercalation. The methods used to study this problem include equilibrium dialysis, relaxation kinetics and hydroxdynamic studies on low‐molecular‐weight and closed‐circular DNA. These show that binding produces no detectable lengthening or unwinding of the double helix, and that the binding reaction is much faster than is the case with unsubstituted proflavine. We conclude that an outside‐bound complex is formed.

We also measured equilibrium binding isotherms for DNA samples of varying G · C content, and for several synthetic polynucleotides. These results are consistent with a model in which a bound 2,7‐di‐t‐butyl‐proflavine molecule occupies three base pairs, and strong binding requires that two of these three be A · T pairs. Comparison of poly(dA) · poly(dT) with poly(A) · poly(U) shows a preference of at least 100‐fold for binding to the former over the latter. Also, we found that the thymine methyl groups are probably not responsible for the A · T specificity.

The optical properties of the outside complex include a red shift of the absorbance maximum, with an increase in maximum absorbance instead of the more common decrease. The fluorescence of the dye is enhanced on binding, especially to an A · T‐rich‐DNA.

The kinetic results show that two complex forms are present, differing in intrinsic affinity by about an order of magnitude. Both are formed with second‐order rate constants in excess of 108 M−1 s−1 and it is therefore very unlikely that either is an intercalated form. The absorbance spectrum of the bound dye changes as the degree of binding increases, due, at least in part, to the changing contribution of the two binding mechanisms. The conclusions concerning binding specificity refer to the dominant complex form; we have no firm information about the specificity of the weaker complex.
On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl‐tRNA Synthetases
Authors: Pachmann Ulrich , Cronvall Eva , Rigler Rudolf , Hirsch Reinhard , Wintermeyer Wolfgang , Zachau Hans G.
Published: March 2005

The interactions of yeast seryl‐tRNA and phenylalanyl‐tRNA synthetases with the cognate and the noncognate tRNA have been studied by fluorescence spectroscopy. The binding process was followed by observing the changes in tryptophan fluorescence of the synthetases as well as the changes in the polarized fluorescence of the tRNAs covalently labeled with ethidium bromide, proflavine, or 1, N6‐etheno‐ATP. The degree of polarization of the fluorescence labels is particularly sensitive to changes in rotational diffusion of the tRNA. Both methods can be regarded as complementary and gave comparable results. There exist, however, cases such as the interaction between seryl‐tRNA synthetase and tRNASer in the absence of Mg2+, where the binding process could only be detected by measurement of the fluorescence polarization.

Unspecific interactions between synthetases and tRNAs (or tRNA‐dye compounds) could be detected and were found to be highly dependent on pH, salt concentration and on the buffer used. In potassium phosphate buffer pH 7.3 discrimination between cognate and noncognate tRNA by seryl‐tRNA synthetase increases with addition of salt. The tryptophan fluorescence of phenylalanyl‐tRNA synthetase was influenced differently by the cognate and the noncognate tRNA while the stabilities of the complexes between this synthetase and the ethidium‐labeled tRNAPhe and tRNASer were rather similar. Seryl‐tRNA synthetase apparently discriminates more efficiently than phenylalanyl‐tRNA synthetase between the cognate and the noncognate tRNAs.

The presence of two binding sites on seryl‐tRNA synthetase and the presence of one binding site on phenylalanyl‐tRNA synthetase for cognate as well as noncognate tRNA could be deduced. For the specific interaction between tRNASer and seryl‐tRNA synthetase in the presence of Mg2+ indications for cooperative behavior have been found. Experiments on competition between noncognate and cognate tRNAs as well as tRNA half molecules for the binding to the synthetase indicate an overlap of specific and unspecific binding sites.

We conclude that the unspecific interaction may be an important initial step preceding the specific binding and recognition of the tRNA by the synthetase.
The Time Dependence of the Activity of δ‐Chymotrypsin at High pH
Authors: Garel Jean‐Renaud , Labouesse Bernard
Published: March 2005

When δ‐chymotrypsin at a high initial pH is mixed with a specific ester substrate, the hydrolytic reaction exhibits a lag phase that can be observed from the release of both products, the alcohol or the amino acid. This lag phase is under the dependence of the ionization of a group of pK 9, at 15°C, which has been previously shown to be the α‐amino group of isoleucine‐16. This lag corresponds to the time needed for the enzyme to isomerize from its “high‐pH” to its “neutral” conformation, and fluorescence measurements have been separately used to monitor this isomerization in the presence of substrate. The initial activity observed when the enzyme is mixed with the substrate is proportional to the concentration of enzyme present in its neutral conformation and is not significantly measurable when the enzyme is in its high‐pH conformation.

It is concluded that the high‐pH form of δ‐chymotrypsin is inactive and cannot be involved in the observed activity of the enzyme at high pH. Therefore the activity of δ‐chymotrypsin exhibited at high pH is linked to a substrate‐promoted isomerization of the protein, that is, to a displacement of the conformational equilibrium presented by the protein towards its active neutral conformation.
Analysis of the Methylation Sites in Yeast Ribosomal RNA
Authors: Klootwijk Jacobus , Planta Rudi J.
Published: March 2005

The methylation sites and the number of methyl groups of 26‐S and 17‐S ribosomal RNA (rRNA) of Saccharomyces carlsbergensis were investigated.

28‐S and 17‐S rRNA contain 43 and 24 methyl groups per molecule, respectively, of which in both molecules 6 are attached to the heterocyclic bases. In 26‐S rRNA the base‐methyl groups are present as two copies of m3U, m5C as well as m1A, whereas the methylated bases in 17‐S rRNA are one m7G, one methylated cytidine and two of m26A.

In 17‐S rRNA there are 11 different alkali‐stable (i.e. ribose‐methylated) dinucleotides, while 26‐S rRNA contains 15 different alkali‐stable dinucleotides, and in addition 4 alkali‐stable trinucleotides (Am‐Cm‐Gp, Am‐Gm‐Cp, Am‐Am‐Up and Um‐Gm‐Ψp).

Most of the methylated oligonucleotides found in a ribonuclease T1 digest of 26‐S or 17‐S rRNA occur in approximately one‐molar amounts. The combined data suggest that yeast ribosomal RNA is essentially homogeneous in its methylated sequences and in addition that the methylation is a highly specific process.
Influence of Buffers, Protein Concentration and Ionic Strength upon the Dissociation of Pila Haemocyanin
Authors: Cox Jos A. , Elliott Francis G.
Published: March 2005

The study of the dissociation of Pila leopoldvillensis haemocyanin by means of light scattering has shown that the protein concentration has no influence upon the dissociation into halves and tenths.

Different buffer ions were shown to have specific effects upon the dissociation of Pila haemocyanin: with maleate and phosphate the dissociation curves are shifted towards lower pH values; with imidazole and Tris the shift is in the opposite direction.

Takening into account the high molecular weight of the protein, the binding of chloride ions, as determined by the ΔHp method, is very small. Therefore pure KCl solutions rather than buffers were used in further work on the dissociation of haemocyanin.

In view of these observations an attempt is made to interpret the influence of the pH and ionic strength upon the dissociation‐association reaction according to a sequential loop model: the forward and back reaction proceed along opposite sequential pathways, each starting with a particular change of the ionisation state of the wholes, halves or tenths. According to this reaction scheme the ionisation constant of the sites responsible for the dissociation into halves is 6.96; and that for the dissociation of halves into tenths is 9.65.
Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates
Authors: Baumann Werner K. , Bizzozero Spartaco A. , Dutler Hans
Published: March 2005

A number of peptide substrates of the general structure Ac‐Lxn‐ … ‐Lx2‐Lx1‐Gly‐NH2 have been synthesized and their α‐chymotrypsin‐catalyzed hydrolyses studied. The acylation rate constants, k23 (=kcat), and the dissociation constants of the enzyme‐substrate complexes, KEA (=Km), have been determined using a modified pH‐stat and a numerical method for the acquisition and processing of the data. On the basis of these constants a quantitative relationship between the peptide structure N‐terminal to the cleaved bond and reactivity has been determined. The results are shown to be consistent with the enzyme‐substrate interaction scheme proposed in 1971 by Segal et al. (Biochemistry 10, 3728). A comparison of the k23/KEA values indicates that the influence of a single structural change on the overall reactivity is virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k23 and KEA values shows that, in general, changes in substrate structure are mainly reflected by changes in k23 rather than in KEA. A few exceptions have been found: KEA or KEA and k23 change when glycine is introduced at the x2 position, when this glycine is replaced by alanine or when alanine is introduced at the x3 position.
Heterogeneity and Complexity of Chlorella Chloroplastic DNA
Authors: Bayen Marcel , Rode André
Published: March 2005

The heterogeneity and complexity of Chlorella chloroplastic DNA has been investigated by means of thermal denaturation and renaturation kinetics.

The chloroplastic DNA presents a Tm of 82.2°C, a maximal hyperchromicity of 40.2% and a dispersion coefficient Δ2/3 of 6°C. The absorbance melting profiles display irregularities indicating an intramolecular heterogeneity of the DNA. The first derivated curves of the melting profiles show the presence in the chloroplastic DNA of two main components of Tm 81 °C and 84.7 °C (which represent together about 80% of the total DNA) and possibly three other minor ones.

Denatured and sheared DNAs renature rapidly as a fast and a slow‐renaturing class with a kinetic complexity of 3 · 107 daltons and 2.3 · 108 daltons respectively (assuming a value of 2.5 · 109 daltons for Escherichia coli DNA). If correction is applied to account for the low d(G + C) content of the chloroplastic DNA, the kinetic complexity of the slow‐renaturing component is found to be in the range of 1.2‐1.3 · 108 daltons.

Our results confirm and extend the fact that chloroplastic DNAs are similar in terms of kinetic complexity and heterogeneity. This is in good agreement with a possible common origin for all the chloroplastic DNAs.

From the discrepancy between analytical and kinetic complexities of Chlorella chloroplastic DNA, one can assume that this DNA is extensively reiterated and that the unique nucleotide sequence is present in about 20 copies in the chloroplastic genome.
Stimulation of ATP Synthesis by a Membrane Potential in Chloroplasts
Authors: Schuldiner Shimon , Rottenberg Hagai , Avron Mordhay
Published: March 2005

Chloroplasts preloaded with protons by light‐induced proton uptake do not synthesize ATP in a following dark period if the proton concentration gradient (ΔpH) is lower than 2.5–3.0 pH units. At such suboptimal ΔpH values synthesis of ATP can be obtained by imposing a diffusion potential across the chloroplast membrane. This potential was realized by providing a high external concentration of either KCl in the presence of valinomycin or of NaCl in the presence of monactin. Diffusion‐potential‐induced stimulation of ATP synthesis was observed both in chloroplasts and in bacterial chromatophores.

The stimulation could be abolished by the addition of a highly permeant anion, like picrate. It is proposed that the effect of the diffusion potential indicates that an increase in the electrochemical gradient of protons can be coupled to the synthesis of ATP.
Precursor–Product Relationship of Encephalomyocarditis Virus‐Specific Polypeptides
Authors: Dobos Peter , Ploude Jean Yves
Published: March 2005

The interrelationship of encephalomyocarditis (EMC) virus‐specific polypeptides was analysed by comparing their [35S]methionine‐labelled tryptic peptides using thin‐layer electrophoresis and chromatography. It was found that the peptide patterns of EMC virus is similar to that of polypeptide A, whereas the fingerprint patterns of polypeptides C, D and E are similar to each other but differ greatly from that of A and purified virus. This is consistent with the proposal that polypeptide A is the precursor of the virus capsid proteins, and that polypeptide E is the cleavage product of D and C. The large number of labelled peptides found in the digests of low‐molecular‐weight proteins G, H, I indicates that they are a mixture of different polypeptides of similar molecular weight generated by the intracellular cleavage of large precursor molecules.
Simulation of Rate‐Zonal Centrifugation on a Digital Computer
Authors: Steensgaard Jens , Funding Lars , Meuwissen Jules A.T.P.
Published: March 2005

A numerical method is described for digital simulation of rate‐zonal centrifugation of particles with known sedimentation and diffusion coefficients in sucrose gradients at given temperatures. A model rotor based on the geometry of a B‐XIV titanium zonal rotor is used. The rotor chamber is divided into 207 annular segments by 206 equidistant cylindrical planes. Initially 207 paired values describing the concentration radius profiles of sample and gradient material together with some physical constants are provided as input data. The net fluxes of material across each plane during a short period of time are calculated. The concentrations in each segment are adjusted accordingly, yielding new concentration radius profiles. New values of density and viscosity in each segment are calculated before the next transfer of materials is allowed to take place. The iterations are continued until a predetermined cumulative run time is reached. The advantages and the drawbacks of this approach are discussed. Comparison with an actual experiment revealed that the simulation method can be used to predict final particle distribution with rather good accuracy. Moreover, the hydrodynamic stability of sample particles can be evaluated at each time point and at each distance during a centrifugation. Hence, this numerical approach can be used to optimize experimental conditions in all types of zonal centrifugation.
Structure‐Activity Relationship in Tryptophanyl‐Transfer Ribonucleic Acid Synthetase from Beef Pancreas
Authors: Iborra François , Mourgeon Gérard , Labouesse Bernard , Labouesse Julie
Published: March 2005

The titration of the – SH groups of beef pancreas tryptophanyl‐tRNA synthetase by N‐ethylmaleimide and the consequent effect on the exchange and transfer reactions of the enzyme were examined.

The 16 sulfhydryl groups of the native dimeric enzyme do not react homogeneously at pH 7, with 1 mM reagent. They can be divided into three classes with decreasing reactivities. The reaction of the first class (6 – SH groups) has no effect on activity. The reaction of the second class (4 – SH groups) brings about an increase of Km(app) for tryptophan both in the exchange and in the transfer reactions of the enzyme. The activity of the enzyme is lost during reaction of the third class (6 ‐ SH groups). This is evidenced by the decrease of the V of the [32P]PPi‐ATP isotope exchange and tRNA aminoacylation reactions and by the decrease in the amount of the tryptophanyladenylate · enzyme complex isolatable on Biogel P30. The destruction of the active site by alkylation of the sulfhydryl groups of class III can be the consequence of a structural change of the enzyme induced by the chemical modification. During the alkylation of the first two classes of ‐ SH groups the Km(app) for ATP, PPi and tRNA are not altered significantly.

The presence of tryptophan or tryptophanyladenylate reduces the rate of titration of the ‐SH groups of class II and class III and in a parallel fashion the rates of increase of Km(app) for tryptophan and of decrease of V. The titration of the –SH groups of class I is not affected by the presence of substrates. The alkylation in the presence of tryptophanyladenylate demonstrates that class II is not homogeneous and that the two molecules of adenylate bound to the dimeric enzyme specifically protect two –SH groups out of the four of this class.
Studies on the Properties of Fish Hemoglobins Molecular Properties and Interaction with Third Components of the Isolated Hemoglobins from Trout (Salmo irideus)
Authors: Brunori Maurizio , Giardina Bruno , Chiancone Emilia , Spagnuolo Carla , Binotti Ines , Antonini Eraldo
Published: March 2005

This paper reports some physico‐chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV).

Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above ∼ 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine.

The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value ofθ at 222 nm becomes more negative by about 5%. On the other hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands.

Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to ∼ one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis.

The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of Hb trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors.
The Pathway of Exogenous and Endogenous Carbohydrate Utilization in Escherichia coli: A Dual Function for the Enzymes of the Maltose Operon
Authors: Palmer T. Norman , Wöber Günter , Whelan William J.
Published: March 2005

The components of the maltose‐utilizing system in Escherichia coli ML, i.e. maltose permease, amylomaltase and maltodextrin phosphorylase, were re‐investigated, with the following findings:

The concentrating mechanism (permease) is the rate‐limiting step in the maltose‐utilizing system. Maltose and higher maltodextrins, up to an average chain length of at least 10 glucosyl units, can be taken up at the same rate. Maltodextrins of an average chain length of 20 glucosyl units are restricted in their uptake, as reflected in the growth‐rate of the organism.

Growth studies with extensively purified maltose show that this sugar, in contrast to maltotriose and higher maltodextrins, is a poor inducer of the system.

Pullulanase is co‐induced by maltodextrins, along with the other enzymes. The pullulanase seems to be bound to the cell wall and has properties very similar to those of the well‐studied pullulanase of Aerobacter aerogenes.

The complete system, now referred to as the maltodextrin‐utilizing system, comprises the enzymes pullulanase, maltodextrin permease, (a name replacing maltose permease) amylomaltase and maltodextrin phosphorylase.

This group of enzymes was also demonstrated to be present in Streptococcus mutans and A. aerogenes. In addition, A. aerogenes possesses an inducible α‐amylase. This last enzyme permits a wider range of branched 1,4‐α‐glucans to be utilized by the latter organism.

The enzymes of glycogen degradation in E. coli NCTC 5928 were investigated. This organism utilizes isoamylase to debranch the reserve polysaccharide, liberating the component chains. These maltodextrin chains are subsequently metabolized by the enzymes of the maltodextrin‐utilizing system.

We suggest that E. coli uses pullulanase for the debranching of extracellular carbohydrates, while isoamylase is presumed to function intracellularly in the degradation of glycogen.

Maltodextrins are provided for intracellular utilization in E. coli by either of two routes. Pullulanase and permease function in the degradation and uptake of extracellular branched α‐glucans. Isoamylase produces maltodextrins during the debranching process of intracellular glycogen. Maltodextrins arising by either route are metabolized to glucose and glucose 1‐phosphate by the concerted actions of amylomaltase and maltodextrin phosphorylase.

A. aerogenes and S. mutans probably have similar enzymic machinery, the former also having an extracellular α‐amylase to assist exogenous glucan degradation.

9. A general scheme for the metabolism of branched 1,4‐α‐glucans in bacteria and plants is proposed.
Inhibition of Stable RNA Synthesis by Levallorphan in Escherichia coli
Authors: Boquet Paul L. , Fromageot Pierre , Devynck Marie‐Aude , Monnier Chantal , Röschenthaler Robert
Published: March 2005

The specific inhibition of stable RNA synthesis induced by levallorphan (N‐allyl‐hydroxy‐morphinane) in Eschericia coli is not permanent but possesses the characteristics of a “shiftdown” regulation pattern similar to those induced by nutrient deficiencey. The transient accumulation of guanosine 3′,5′‐bis(diphosphate) (ppGpp or MS I) caused by levallorphan is provoked mainly by a restriction of the ability of bacteria to degrade this nucleotide. Based on the behavior in vivo of the levels of compound MS II (guanosine 3′‐diphosphate‐5′‐triphosphate or pppGpp) in the presence of the morphinane, a model is proposed according to which MS II would normally be synthesized using MS I as a precursor, in a levallorphan‐sensitive step subsequent to MS I synthesis.
d‐Galactose Dehydrogenase from Pseudomonas saccharophila
Authors: Wengenmayer Friedrich , Ueberschär Karl‐Heinz , Kurz Gerhart , Sund Horst
Published: March 2005

d‐Galactose dehydrogenase from Pseudomonas saccharophila has been purified over 700‐fold with an over‐all yield of 27%. The purified enzyme exhibits a specific activity of about 200 μmol NADH formed × min−1× mg protein−1 and has been shown to be homogeneous by ultracentrifugation and by gel electrophoresis.

The amino acid composition of the enzyme has been determined.

The native enzyme has a sedimentation coefficient (S20,w0) of 5.94 S and a diffusion coefficient (D20,w0) of 5.60 F. The experimentally determined partial specific volume (v̄) is 0.744 ml/g. The molecular weight calculated from these data is 103000, in good agreement with the value of 102000 estimated by gel permeation chromatography.

The enzyme dissociates into polypeptide chains in the presence of 5 M guanidine hydrochloride or 0.1% sodium dodecylsulfate. The molecular weight of the polypeptide chains has been found to be 25000 by ultracentrifugation (S20,w0= 1.35 S, D20,w0= 5.28 F) and by dodecylsulfate‐polycrylamide electrophoresis. The native enzyme is composed of four polypeptide chains.

The pI of d‐galactose dehydrogenase is 5.1. The pH‐optimum is between pH 8–9 in Tris‐HCI buffer. AT PH 8.6 and 30°C the limiting michaelis constant for NAD+ is Ka= 0.14 mM, the limiting michaelis constant for d‐galactose is Kb= 1.0 mM and the dissociation constant for NAD+ is Kia= 0.48 mM.

The enzyme is specific for NAD and uses NADP only to a minor extent as a coenzyme. It oxidizes the following sugars arranged according to relative rates, in decreasing order: d‐FUCOSE > d‐galactose > 3‐deoxy‐d‐galactose > 2‐deoxy‐d‐galactose > 4‐deoxy‐d‐galactose > l‐arabinose > 2‐deoxy‐2‐amino‐d‐galactose.
The Specificity of Pancreatic Deoxyribonuclease
Authors: Ehrlich Stanislav D. , Bertazzoni Umberto , Bernardi Giorgio
Published: March 2005

The kinetics of degradation of calf thymus DNA by pancreatic DNAase has been investigated with the main purpose of providing the background for studying the specificity of the enzyme and its use in investigations of nucleotide sequences in DNA. The relationships between the hyperchromic shift or acid solubility and the reciprocal average degree of polymerization (P̄n−1) of the hydrolysate have been established; these can be used in order to prepare oligonucleotides of desired average chain length simply by measuring the increase of absorbance or of acid solubility of the digest during DNA degradation.

The specificity of the pancreatic DNAase was studied by determining the base compositions of 3′‐terminal, 5′‐terminal and 5′‐penultimate nucleotides of oligonucleotides released by the enzyme. The 3′‐terminal nucleotide composition was found not to vary in the P̄n, or average size, range 150 to 8 and 30 to 8 for digestions of calf thymus DNA in the presence of magnesium and manganese ions, respectively, indicating that no change in the specificity takes place during digestion. The compositions of 3′‐terminal, 5′‐terminal and 5′‐penultimate nucleotides differ from those expected on the basis of random splitting of DNA. The conclusion is drawn that pancreatic DNAase recognizes sequences at least three nucleotides long.
Rate‐Limiting Factors in Ethanol Oxidation by Isolated Rat‐Liver Parenchymal Cells
Authors: Grunnet Niels , Quistorff Bjørn , Thieden Herluf I. D.
Published: March 2005

Isolated rat‐liver parenchymal cells oxidized ethanol at a rate of 1.4, 1.7, 1.9 and 2.5μmol/min per ml packed cells at 4, 20, 40 and 65 mM ethanol, respectively. Between 40 and 65 mM ethanol an abrupt 30% increase in the ethanol oxidation rate was observed.

The activity of the NAD‐dependent alcohol dehydrogenase in a homogenate of isolated cells corresponded to 1.25–5 U/ml packed cells, depending on the assay method used.

Fructose or pyruvate enhanced the oxidation rate of ethanol by 1.4–2.0 μmol/min per ml packed cells independent of the ethanol concentration applied. No additive effect of the two compounds upon ethanol oxidation was observed.

Pyrazole inhibited the NAD dependent alcohol dehydrogenase activity in a homogenate of isolated cells as well as the unstimulated and the fructose‐stimulated ethanol oxidation with a Ki‐value of 9–13 μM.

The “fructose effect” was inhibited 100% by pyrazole concentrations, which inhibited the unstimulated ethanol oxidation only 30%, indicating that the “fructose effect” is mediated via alcohol dehydrogenase.

mM pyrazole only slightly inhibited that part of the ethanol oxidation which was not catalyzed by alcohl dehydrogenase, whereas 18 mM pyrazole inhibited also this pathway significantly.

50 μ pyrazole had no effect at all upon the basal ethanol oxidation rate wheras the fructose‐stimulated ethanol oxidation was 30% inhibited.

The results concerning the effect of fructose and pyruvate upon ethanol oxidation are interpreted in terms of the mechanism for the reaction catalyzed by alcohol dehydrogenase. At low concentrations of ethanol and in the absence of fructose, the rate‐limiting step in ethanol oxidation appears to be dissociation of the enzyme‐NADH complex, whereas, in the presence of fructose, the maximal activity of ethanol dehydrogenase may be rate‐limiting for the oxidation of ethanol.

The results also suggest that enzyme systems other than alcohol dehydrogenase participate in ethanol oxidation at high concentrations of ethanol.
Binding of the Initiation Factor IF‐3 to Escherichia coli Ribosomes and MS2 RNA
Authors: Vermeer Cees , Boon Jan , Talens Anneke , Bosch Leendert
Published: March 2005

The binding of IF‐3 to 70‐S, 50‐S and 30‐S ribosomes and subunits of Escherichia coli and to MS2 RNA was studied employing Sephadex G‐200 chromatography and sucrose‐gradient centrifugation to recover the complex formed. In the latter case, reaction mixtures were fixed with 0.25% glutaraldehyde prior to centrifugation. Total reaction mixtures were also analyzed by polyacrylamide‐gel electrophoresis.

Highly purified IF‐3, either unlabeled or labeled with 35S was employed and consistent results were obtained with both factor preparations.

Binding of IF‐3 to 30‐S subunits was independent of the Mg2+ concentration in the range 1–14 mM. The binding to 50‐S subunits and MS2 RNA was counteracted by increasing the Mg2+ concentration in the same range.

Binding at 0°C and at 37°C occurred to the same extent both with 30‐S and 50‐S ribosomal subunits.

The number of IF‐3 molecules bound per 30‐S subunit approached unity, that bound per 50‐S subunit exceeded unity when the IF‐3 input was sufficiently raised.

At low factor/ribosome input ratios IF‐3 binds preferentially to 30‐S particles. When the ratio is increased binding to 50‐S subunits and 70‐S ribosomes also occurs. The IF‐3‐mediated dissociation of 70‐S ribosomes is strongly inhibited by adding increasing amounts of 30‐S subunits.

At all Mg2+ concentrations studied the relative extent of binding can be expressed as: 30‐S > 70‐S, 50‐S > MS2 RNA.

Binding to all types of ribosomes and subunits and to MS2 RNA is not stimulated by the addition of IF‐1 and IF‐2. The latter factors stabilize the 30‐S · IF‐3 complexes, however.