bluuebunny/crossref_metadata_2025 huggingface dataset:
The Influence of 6‐M Urea on 30‐S Ribosomes of Escherichia coli
Authors: SPITNIK‐ELSON Pnina , GREENMAN Benjamin , ABRAMOVITZ Renne
Published: March 2005

When Escherichia coli 30‐S ribosomes are treated with 6 M urea in the presence of Mg2+, their sedimentation coefficient drops to 16–17 S, and their circular dichroism spectrum changes in a way that indicates a change in the conformation of the proteins in the ribosome. Under these conditions proteins are detached by relatively low concentrations of salt, NH4Cl and magnesium acetate being comparably effective. The proteins remaining attached to the RNA at different ionic strengths have been identified. It is concluded that the conformation of the proteins in situ in the ribosome is a determining factor affecting the structure of the ribosome and the detachment of the proteins by salt.
Processing and Release o Ribosomal RNA from Isolated Nuclei Analysis of the ATP‐Dependence and Cytosol‐Dependence
Authors: RACEVSKIS Janis , WEBB Thomas E.
Published: March 2005

The release of ribosomal subunits from isolated liver nuclei is stimulated by macromolecules in the cytosol, the concentration‐dependence curve being sigmoidal in character. These factors appear to be proteins which adsorb to DEAE‐cellulose at pH 8.0, precipitate between 20 and 60% saturation with ammonium sulfate and are partially denatured during a short incubation at 50 °C, or during prolonged storage at ‐15 °C. Pre‐incubation of the nuclei at 30 °C for 10 min prior to the addition of ATP neither resulted in release of RNA, nor affected the subsequent rate of release when the energy source was added. The degree of processing of the 45‐S ribosomal precursor RNA was also directly or indirectly dependent on the presence in the incubation medium of ATP and cytoplasmic macromolecules.
Direct Fluorometric Determination of Liver Alcohol Dehydrogenase with Berberine
Authors: KOVÁŘ Jan
Published: March 2005

A fluorometric method for the determination of the concentration of horse liver alcohol dehydrogenase is described. It is based on the formation of a strongly fluorescent complex between the enzyme and berberine. The relative increase of fluorescence after the addition of the enzyme into a solution of berberine (at fixed concentration) is a linear function of the enzyme concentration.

The results are easy to obtain, nearly temperature independent and as reliable as those of the commonly used activity measurement.

The principle of this method can also be used for the estimation of other proteins forming strongly fluorescent complexes with slightly fluorescent probes.
Regulatory Behavior of Monomeric Enzymes
Authors: RICARD Jacques , MEUNIER Jean‐Claude , BUC Jean
Published: March 2005

A new concept, that of a mnemonical transition, is proposed to explain the departure from Michaelian behavior of monomeric enzymes following ordered reaction mechanisms. The concept integrates three simple ideas: the free enzyme occurs under two conformational states in equilibrium; the collision of any of these forms with the first substrate induces the same third new configuration required for proper substrate binding; the collision of only one of these enzyme forms with the last product stabilizes that form without any new conformational change. This whole set of definitions is equivalent to assuming that the free enzyme which is released after catalysis, is in a conformation different from the initial one. The enzyme can be said to “recall” for a while the configuration stabilized by the last product before relapsing to the initial conformation. The non‐hyperbolic behavior is thus the consequence of the cooperation of two different conformations of the free enzyme to the overall reaction process.

The reciprocal steady‐state rate equations have been established and thoroughly discussed both for one‐substrate, one‐product and two‐substrate, two‐product mnemonical enzymes. The departure from Michaelian behavior does not appear as a consequence of a slow conformational transition, but is defined in a simpler way by the relative values of the activation free energies of conformation changes required for substrate binding on the two enzyme forms.

A two‐substrate, two‐product enzyme following an ordered reaction mechanism and exhibiting the mnemonical transition has a very distinctive kinetic behavior. The curvature of the primary plots is observed with regard to the first substrate only, and is independant of the concentration of the second substrate as well as that of the first product. The enzyme is not inhibited by an excess of the substrate and the primary plots are either concave up or down. The slopes and the intercepts of the straight lines obtained in double reciprocal plots with the second substrate should give, when these are replotted against the reciprocal of the first substrate concentration, a straight line and a curve, respectively. The cooperation of the enzyme conformations to the overall reaction process can be either positive or negative. Since the reciprocal plots cannot exhibit an extreme, the extent of that cooperation can be measured by the numerical value of the second derivative of the reciprocal rate equation. The extent of cooperation between the free enzyme forms is highly controlled by the concentration of the last product. If the cooperation was already negative, the product strengthens that cooperation. If, on the other hand, the cooperation was positive, the product decreases or even reverses that cooperation.

A very general property of one‐sited mnemonical enzymes is that cooperation between enzyme forms is only kinetic and does not appear in the substrate binding isotherms.
Quantitation of Ovalbumin mRNA in Hen and Chick Oviduct by Hybridization to Complementary DNA
Authors: COX Ronald F. , HAINES Michael E. , EMTAGE J. Spencer
Published: March 2005

DNA complementary to ovalbumin mRNA was synthesized using reverse transcriptase from avian myelobastosis virus and ovalbumin mRNA, purified 60–70‐fold with respect to polysomal RNA, as template.

The quantitative distribution of ovalbumin mRNA sequences in hen and chick oviduct was determined by hybridization with ovalbumin complementary DNA (cDNA). In hen, we calculate that there are approximately 36000 ovalbumin mRNA molecules per oviduct tubular gland cell. The polysome fraction contains 93% of ovalbumin mRNA; nuclear and non‐polysomal cytoplasmic RNA fractions contain 5% and 2% respectively. In the nucleus, the majority of sequences exist as the 16‐S “translatable” form when analyzed on formamide gradients. High‐molecular‐weight RNA species (> 30 S) were detected, and contained less than 2% of total nuclear mRNA sequences.

The appearance of ovalbumin mRNA sequences in total, nuclear and polysomal RNA fractions from chick oviduct was measured in response to estradiol. Prior to hormone treatment, low levels of sequences were present in all fractions, approximately one mRNA molecule per gland cell being detected in nuclei. Up to 2 h after treatment a small increase in ovalbumin mRNA concentration was observed in nuclear and, to a lesser extent, in polysomal RNA. Between 3 and 6 h ovalbumin mRNA accumulates rapidly in the polysome fraction and after 9 h the rate of appearance of this mRNA is approximately 7 molecules per min per gland cell.

Our results indicate that the induction by estradiol of ovalbumin synthesis in oviduct is mediated either by the synthesis of new ovalbumin gene transcripts or the stabilization of ovalbumin gene products.
Yeast Phenylalanyl‐tRNA Synthetase
Authors: FASIOLO Franco , EBEL Jean‐Pierre
Published: March 2005

Phenylalanyl‐tRNA synthetase from yeast forms an enzyme · phenylalanyl adenylate complex, which has been isolated from the reaction mixture by gel filtration. The study of this complex leads to the conclusion that there are two binding sites for phenylalanyl adenylate per molecule of enzyme.

No complex between enzyme and ATP could be characterised. Mg2+ is required for complex formation, but it can be replaced by monovalent cations in the transfer of phenylalanine from the complex to tRNAPhe. The kinetics of the transfer reaction show evidence that the rate‐limiting step is not the release of enzyme‐bound Phe‐tRNAPhe, but the acylation of tRNAPhe after the ternary complex phenylalanyl adenylate · enzyme · tRNAPhe has been formed.
The Distribution of Histones and Nonhistone Proteins in the ECTHAM‐Cellulose Fractions of Chromatin from Several Tissues
Authors: REECK Gerald R. , SIMPSON Robert T. , SOBER Herbert A.
Published: March 2005

Chromatins from rabbit kidney and brain, calf liver, HeLa cells, and duck erythrocytes have been subjected to chromatography on ECTHAM‐cellulose, which has previously been shown to fractionate rabbit liver chromatin into low and high‐melting nucleoprotein species [Reeck et al. (1972) Proc. Natl Acad. Sci. U.S.A. 69, 2317–2321].

Except for duck erythrocyte chromatin, the late‐eluted low‐melting chromatin fractions were greatly depleted in histone F1, as we reported previously for rabbit‐liver chromatin. No differences were found in the proteins of the ECTHAM‐cellulose fractions of duck‐erythrocyte chromatin, which, in contrast to all other chromatins examined, is known to be inactive in RNA synthesis. Differences in the nonhistone proteins of the early and late‐eluted chromatin fractions have been observed which appear to be unique for chromatin from each source.
Patulin Biosynthesis: The Role of Mixed‐Function Oxidases in the Hydroxylation of m‐Cresol
Authors: MURPHY Gillian , VOGEL Günter , KRIPPAHL Günther , LYNEN Feodor
Published: March 2005

Two hydroxylation reactions of m‐cresol, an intermediate in the secondary biosynthesis of the antibiotic patulin, have been characterised in cell‐free preparations of Penicillium patulum. The ability to convert m‐cresol to 2,5‐dihydroxytoluene and m‐hydroxybenzyl alcohol is located in separable fractions of the 105000 × g microsomal pellet. The activities had Km values for m‐cresol of 80 μM and 5 μM, respectively. Both reactions require molecular oxygen and NADPH for activity. Inhibition of m‐cresol hydroxylases by carbon monoxide is reversible by light with a photochemical action spectrum maximal at 450 nm. Carbon monoxide difference spectra of mycel extracts show the presence of absorbing pigments at 422 and 450–455 nm.

The enzymes are not appreciably inhibited by cyanide but are inhibited by cytochrome c. m‐Cresol hydroxylation therefore appears to be catalysed by typical mixed‐function oxidases involving an NADPH‐dependent reductase and a pigment like cytochrome P‐450.

In the light of studies on the utilisation of radioactively labelled intermediates, the methyl hydroxylation of m‐cresol to m‐hydroxybenzyl alcohol probably represents an important reaction in the sequence leading to patulin formation. Ring hydroxylation of m‐cresol to 2,5‐dihydroxytoluene appears to be a side reaction functional at higher concentrations of m‐cresol.
Role of Histones in Chromatin Condensation
Authors: BILLETT Michael A. , BARRY J. Michael
Published: March 2005

We have attempted to discover which of the proteins of hen erythrocyte chromatin maintain it in the condensed state at physiological concentrations of cations. After removal of histones F2C and F1 from the chromatin there was a change in its sedimentation properties over a range of cation concentrations, and it now resembled an “uncondensed” fraction from rat liver chromatin. Removal of larger quantities of other histones (F2a2, F2b and part of F2al and F3) did not produce this change. On removal of histones F1, F2a2, F2b and F2c condensation of the chromatin was lost. Condensation was regained on reassociation with all the histones removed, or with F1 or F2c, but not with F2a2 plus F2b in similar quantities. Thus histones F2c (peculiar to nucleated erythrocytes) and F1 appear to be primarily responsible for maintaining hen erythrocyte chromatin in a condensed state. We conclude that in the genetically active uncondensed fraction of rat liver chromatin the condensing effect of F1 histones is antagonised by additional negatively charged molecules.
Control of Ornithine Carbamoyltransferase Activity by Arginase in Bacillus subtilis
Authors: ISSALY Inda M. , ISSALY Abel S.
Published: March 2005

Partially purified Bacillus subtilis ornithine carbamoyltransferase is cooperatively inhibited by excess of ornithine. The inhibition is decreased by lowering of pH from 9.2 to 7.6 without altering the affinity of the enzyme for the neutral species of ornithine which seems to be its true substrate.

Arginine and lysine partially reverse this inhibition without affecting the catalytic activity of the enzyme. 2‐Aminobutyrate, an inhibitor of the enzyme, competitive with respect to ornithine, is not able to replace ornithine in this excess‐substrate type of inhibition. Zn2+ is also an inhibitor of ornithine carbamoyltransferase, with competitive action towards ornithine and its inhibition is added to the excess ornithine inhibition. These data suggest that ornithine carbamoyltransferase has a regulatory site for ornithine, distinct from the catalytic one.

Partially purified arginase, prepared from the same strain, increases the inhibition of ornithine carbamoyltransferase by excess of ornithine when arginine is present. An association of the two enzymes is suggested by molecular sieving. These data postulate that ornithine carbamoyltransferase is regulated by arginase under the control of arginine and ornithine. The other substrate, carbamoyl phosphate, is not involved in this regulatory mechanism.

Lysine and Zn2+ inhibit arginase competitively with respect to arginine without altering the effect of this enzyme on the inhibition of ornithine carbamoyltransferase. 2‐Aminobutyrate inhibits arginase non‐competitively with respect to arginine and prevents the inhibition of arginase towards ornithine carbamoyltransferase. These results suggest that arginase possesses a regulatory site for arginine, distinct from the catalytic one, which is also involved in the control mechanism.
Utilization of the Carbon and Hydrogen Atoms of Ethanol in the Biosynthesis of Steroids and Bile Acids
Authors: CRONHOLM Tomas , BURLINGAME Alma L. , SJÖVALL Jan
Published: March 2005

[1‐2H2]Ethanol, [2‐2H3]ethanol and [1‐13C, 1‐2H2]ethanol were administered to bile fistula rats under conditions ensuring a maximal rate of oxidation for about 24 h. Cholesterol, cholic acid, chenodeoxycholic acid and 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one were isolated from bile collected at intervals during the experiment. The fraction of molecules containing different numbers of heavy isotope atoms was measured by gas chromatography–mass spectrometry–computer techniques. The results were used to determine the origin of the cholesterol pools used as precursors for the different compounds isolated from bile.

The bile acids were found to be formed from a cholesterol pool, the turnover of which was about 5 times more rapid than that of the cholesterol pool used for biliary excretion. The cholesterol pools used for biliary excretion and as corticosterone precursor seemed to be in equilibrium.

The acetyl groups derived from ethanol were introduced into the steroids without exchange of hydrogens at C‐2, and the acetyl‐CoA pool used as precursor did not exchange with acetyl groups in e.g. citrate or fatty acids. A difference in incorporation of [2‐2H3]acetyl groups and [1‐13C]acetyl groups was interpreted to be due to an isotope effect. Ethanol contributed about 45% of the acetyl‐CoA pool used as precursor of cholesterol and bile acids.

Deuterium from [1‐2H2]ethanol was incorporated in many positions of all the steroids. If it is assumed that the incorporation is mediated by NADPH, the deuterium excess of the hydrogens introduced was about 10 atoms%. The contribution via different pathways of an important part of the hydrogen and carbon atoms in cholesterol, bile acids and corticosteroids provides possible mechanisms by which ethanol may affect the biosynthesis and metabolism of steroids.
Studies on Phenylalanine‐Specific Transfer Ribonucleic Acid from Chick Embryos
Authors: WITTIG Burghardt , REUTER Stephanie , GOTTSCHLING Hubert
Published: March 2005

The phenylalanine‐specific tRNAPhe from 13‐day‐old chick embryo muscle was purified. Its fluorescent and hydrophobic Y+ base was excised by mild acid treatment and characterized chromatographically and by its ultraviolet absorption and fluorescent spectra. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe was characterized.

Chick embryo tRNAPhe looses by the removal of the Y+ base its phenylalanine acceptor capacity.

In this state it has a reduced sedimentation coefficient of 2.8 S, a low Tm value (22°C), only rudimentary hyperchromicity, but unchanged electrophoretic properties.

The compound obtained after removal of the Y+ base from chick embryo tRNAPhe regains normal melting behaviour and phenylalanine acceptor capacity when exposed to special renaturation conditions.

The Km, of the renatured form is 0.25 × 10−6 M and V= 220 pmol × min−1× ml−1, as compared to Km, = 0.11 × 10−6 M and V= 370 pmol × min−1× ml−1 for the native chick embryo tRNAPhe.
Effect of Distamycin A on T4‐DNA‐Directed RNA Synthesis
Authors: PUSCHENDORF Bernd , BECHER Horst , BÖHLANDT Dietmar , GRUNICKE Hans
Published: March 2005

Distamycin inhibits the transcription of T4‐DNA by Escherichia coli RNA polymerase. The inhibition of RNA synthesis is predominantly due to a decrease in RNA chain initiation. The average chain length of the RNA which is synthesized in the presence of the antibiotic remains unaltered up to a ratio of 1 mole distamycin per 30 nucleotides of the DNA. The impairment of the initiation of RNA synthesis is explained by an inhibition of the binding of the RNA polymerase to the DNA. The binding of the RNA polymerase holoenzyme to DNA is affected to a much greater extent than the binding of the core enzyme. A preincubation of RNA polymerase holoenzyme and DNA at 30°C renders the RNA synthesis resistant to the drug. Preincubation of core enzyme and DNA is without any effect. Based on these results it is concluded that distamycin predominantly affects the binding of RNA polymerase to the specific binding sites.
Precursors of Cytochrome Oxidase in Cytochrome‐Oxidase‐Deficient Cells of Neurospora crassa
Authors: WERNER Sigurd , SCHWAB Andreas J. , NEUPERT Walter
Published: March 2005

Three different cell types of Neurospora crassa deficient in cytochrome oxidase were studied: the nuclear mutant cni‐1, the cytoplasmic mutant mi‐1 and copper‐depleted wild‐type cells.

The enzyme‐deficient cells have retained a functioning mitochondrial protein synthesis. It accounted for 12–16% of the total protein synthesis of the cell. However, the analysis of mitochondrial translation products by gel electrophoresis revealed that different amounts of individual membrane proteins were synthesized. Especially mutant cni‐1 produced large amounts of a small molecular weight translation product, which is barely detectable in wild‐type.

Mitochondrial preparations of cytochrome‐oxidase‐deficient cells were examined for precursors of cytochrome oxidase. The presence of polypeptide components of cytochrome oxidase in the mitochondria was established with specific antibodies. On the other hand, no significant amounts of heme a could be extracted.

Radioactively labelled components of cytochrome oxidase were isolated by immunoprecipitation and analysed by gel electrophoresis. All three cell types contained the enzyme components 4–7, which are translated on cytoplasmic ribosomes. The mitochondrially synthesized components 1–3 were present in mi‐1 mutant and in copper‐depleted wild‐type cells. In contrast, components 2 and 3 were not detectable in the nuclear mutant cni‐1. Both relative and absolute amounts of these polypeptides in the enzyme‐deficient cells were quite different from those in wild‐type cells.

The components of cytochrome oxidase found in the enzyme‐deficient cells were tightly associated with the mitochondrial membranes.

Processes, which affect and may control the production of enzyme precursors or their assembly to a functional cytochrome oxidase are discussed.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCHÄFER Rolf , HINNEN Rosmarie , FRANKLIN Richard M.
Published: March 2005

The lipid‐containing bacteriophage PM2 has four distinct structural proteins, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate or electrophoresis on cellulose acetate strips in the presence of 6–8 M urea. The molecular weights of these four polypeptides determined in several polyacrylamide gel systems are: I = 43000, II = 26000–27000, III = 12500, IV = 4700. The isoelectric points of the four viral proteins were determined on disrupted virus particles by electrophoresis on cellulose acetate strips in the presence of 6 M urea at various pH values. The mobilities were extrapolated to zero mobility, a method preferable to isoelectric focusing where the denatured viral proteins aggregate and precipitate near the isoelectric point. The method is well suited for the determination of the isoelectric point of proteins in the native or only partially denatured state. Whereas proteins I, III and IV have isoelectric points slightly under neutrality (pH 6.2, 5.8 and 5.5, respectively), the isoelectric point of protein II is around 12.3. Calcium ions are tightly bound to protein II. After removal of the calcium ions in the presence of 6 M guanidine hydrochloride and EDTA, the pI of protein II is 9. The isoelectric point of the intact virus, determined on cellulose strips, is around 7.3. Protein I, the spike protein, is the only structural polypeptide which is soluble in aqueous solutions around pH 7. All of the proteins are soluble in 6–8 M urea or 6 M guanidine hydrochloride at pH 7–8.5. Proteins III and IV, the nucleocapsid core proteins, have the solubility properties of proteolipids or highly lipophilic polypeptides as shown by their solubility in nonionic detergents and by their distribution in a Bligh and Dyer fractionation. Neither glycolipids nor glyco‐proteins are present in the virus. The distribution of phospholipids has been determined by chemical modification of the outer lamella or outer and inner lamellae of the bacteriophage lipid bilayer. The viral lipid bilayer is asymmetric; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethanolamine residues the inner lipid layer.

A complete model of the virus, including the assembly process, is discussed at the end of this report.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCANDELLA Carl J. , SCHINDLER Hansgeorg , FRANKLIN Richard M. , SEELIG Joachim
Published: March 2005

Motion of acyl chains in the phospholipid bilayer of bacteriophage PM2 was studied by the technique of electron spin resonance. The spin labels used were derivatives of stearic acid with the nitroxide radical in several different positions. The spectra were measured over the temperature range of 0°C to 60°C, within which the spectral changes were reversible. Spectral analysis by means of pertubation theory was carried out over the temperature range of 30°C to 60°C. The spectra, characteristic of a fluid bilayer, indicated that the hydrocarbon chains are in a state of rapid motion; close to the polar region there is a highly ordered environment in which motion is restricted, whereas the bilayer interior is disordered; the strong temperature dependance of the order parameter S3, in the absence of a phase change, and the behaviour of the hyperfine splitting constant aN, suggest that there are strong lipid‐protein interactions.
Effect of 17β‐Estradiol on the Synthesis of Non‐Histone Nuclear Proteins in Chick Liver
Authors: DIERKS‐VENTLING Christa , JOST Jean‐Pierre
Published: March 2005

At 2 h following the injection of 17β‐estradiol into immature chicks, the rate of [3H]leucine incorporation into total nuclear protein of liver, after correction for the free leucine pool, was increased 3.6‐fold over that of the controls. By means of a double‐labelling technique it could be shown in addition, after basic and acidic nucleoproteins were extracted from highly purified liver nuclei, that the stimulation by 17β‐estradiol in vivo resulted within 3 h in the selective synthesis of two non‐histone nuclear proteins of molecular weights of 26000 and 20000. The latter exhibited multiple isoelectric points of 5.0, 5.35 and 6.2.
The Genomic Complexity of Acanthamoeba castellanii Mitochondrial DNA
Authors: BOHNERT Hans J. , HERRMANN Reinhold G.
Published: March 2005

DNA of DNase‐treated mitochondria of Acanthamoeba castellanii bands at a buoyant density of 1.690 g/cm3. Up to 80% of the molecules were circular, with a circumference of 12.7 μm. Two circular conformations, the closed twisted and the open form, were separated in preparative CsCl–ethidium bromide gradients.

When mtDNA was renatured at a temperature Tm– 23°C, deviations from second‐order kinetics were observed. At Tm– 10°C, however, it renatured as a single kinetic class without indications of sequence repetition. The coincidence of the kinetic complexity (2.57 × 107) and the circle contour length within the limits of error of the methods suggests that each circular molecule represents a chondriome.

The main DNA component, which is of nuclear origin and has a peak density of 1.716 g/cm3 in CsCl, displays heterophasic renaturation kinetics. Its slow‐reassociating fraction shows a kinetic complexity corresponding to a molecular weight of 2.1 × 1010. Approximately 22% of the main component appears to be repetitive at a renaturation temperature of Tm– 23°C.
Nuclear‐Magnetic‐Resonance with 13C and 31P and Circular‐Dichroism Studies of a Ternary Complex of Spermine, Cu2+ and AMP
Authors: WESER Ulrich , STROBEL Gert‐Joachim , RUPP Heinz , VOELTER Wolfgang
Published: March 2005

The formation of a ternary complex using cupric ions, basic amines of biological significance, and nucleotides was examined employing high‐resolution nuclear magnetic resonance (NMR) with 13C and 31P and circular dichroism. The reaction of Cu2+ with each ligand was studied separately and as the ternary complex. From 13C‐NMR data the assignment of the respective carbon atoms adjacent to a copper‐binding atom was clearly shown. It is intriguing to note that even in such a ternary complex the N‐7 of the purine ring, as well as the phosphate residue (measured by 13P‐NMR spectroscopy) provide the preferred binding sites for the cupric ion. Marked interactions of the chelated cupric ion with the ribose moiety in the ternary complex were deduced from circular dichroic measurements. No such reactivity was seen when dAMP was employed. The existence of a ternary Cu · AMP · amine complex opens a number of new aspects regarding the reactivity of metal ions in the biochemistry of nucleic acids.
On the Conformation of Pyridoxal Phosphate Imine in Solution and in Aspartate‐Aminotransferase Active Site
Authors: TUMANYAN Vladymir G. , MAMAEVA Olga K. , BOCHAROV Alexander L. , IVANOV Valery I. , KARPEISKY Marat Y. , YAKOVLEV Gennady I.
Published: March 2005

The work comprises:

Determination by nuclear magnetic resonance method of the conformation of the imino group of pyridoxal‐P oxime in solution. It has been found to be nearly coplanar with the aromatic ring.

Theoretical calculation by the method of atom‐atom potentials of the permitted conformations of pyridoxal‐P imine and its analogs: 6‐methylpyridoxal‐P and 5′‐methylpyridoxal‐P. There have been found seven favourable conformations for pyridoxal‐P imine, some of which were prohibited in the analogs mentioned.

Experimental estimation of the conformation of pyridoxal‐P imine in the active site of aspartate aminotransferase with the use of the above sterically hindered analogs and 5′‐deoxy‐5′‐carboxy‐methylenepyridoxal analog. In result, the number of the possible conformations for pyridoxal‐P imine in the active site has been reduced to a pair of symmetrical conformations.
Fluidity in Hydrophobic Protein Regions of Synaptic Membranes
Authors: HOSS Wayne , ABOOD Leo G.
Published: March 2005

Dynamic quenching of native tryptophan fluorescence has been used to assess the fluidity of intact membrane suspensions. The photophysical quenching of tryptophan fluorescence by chloroform occurred by a diffusion‐controlled process where one‐half of all collisions were effective. The bimolecular rate constant for quenching was directly proportional to the fluidity for a series of solvents. Estimation of the rate constant in the membranes from quenching data indicated that the viscosity of membrane proteins (about 1 cP) was at least an order‐of‐magnitude lower than the values reported for lipid bilayer regions of nerve membranes.
Separation of the Proteins in Human Tonsillar Cytoplasmic Ribosomes by Two‐Dimensional Polyacrylamide‐Gel Electrophoresis
Authors: ÜÇLER Ugur , BERMEK Engin
Published: March 2005

Proteins of cytoplasmic ribosomes from human tonsillar lymphatic tissue were analyzed by two‐dimensional polyacrylamide‐gel electrophoresis. 34 proteins were found to be associated with the 40‐S subunit and 35 proteins with the 60‐S subunit. 65 proteins had different electrophoretic mobilities. Two proteins found in the small subunit (S10 and S20) co‐migrated with two large subunit proteins (L13 and L20 respectively). A comparison of our results with those obtained by the same method for ribosomal proteins from human placenta, rat liver and rabbit reticulocytes suggested a possible identity of the electrophoretic mobilities of 21–24 40‐S subunit proteins and 25–26 60‐S subunit proteins.
Catabolism of Hamamelose
Authors: THANBICHLER Alfred , BECK Erwin
Published: March 2005

Hamamelose (2‐C‐hydroxymethyl‐d‐ribose) is a common sugar of green land plants. However, until now, all experiments failed to show dissimilation of this sugar by its producers or by soil microorganisms. For investigation of its biological degradation, microorganisms were used which had been isolated from plant material (leaves of Primula clusiana Tausch). These microorganisms belong to the Enterobacteriaceae and are almost identical to those described by Asai et al. (1956, 1962) as Kluyvera citrophila. They differ from the ATCC strains of this organism by their ability to grow on hamamelose.

Isotope studies using 14C‐labelled hamamelose and glucose (for comparison) as substrates were carried out to elucidate the biochemical route of hamamelose catabolism. This degradation leads to CO2 under aerobic conditions and to lactate, succinate, formate, acetate and ethanol under anaerobic ones. Glucose dissimilation proceeds via glycolysis and leads to the same end‐products. As intermediates of the biological degradation of hamamelose, hamamelose 21‐phosphate, fructose 1‐phosphate, fructose 1, 6‐bisphosphate and phosphoglycerate were identified. Thus the biochemical route of hamamelose dissimilation appears to be rather similar to glycolysis. The following sequence is proposed: hamamelose → hamamelose 21‐phosphate → fructose 1‐phosphate → fructose 1, 6‐bisphosphate → phosphoglycerate → pyruvate → volatile end‐products. Differences from the glycolytic pathway occur at step 2 in which there is a rearrangement of the carbon skeleton and at step 3, in which fructose 1‐phosphate instead of fructose 6‐phosphate has to be phosphorylated.
Interaction between β‐Lactam Antibiotics and Exocellular dd‐Carboxypeptidase‐Transpeptidase of Streptomyces R61
Authors: FRÈRE Jean‐Marie , LEYH‐BOUILLE Mélina , GHUYSEN Jean‐Marie , PERKINS Harold R.
Published: March 2005

On the basis of steady‐state kinetics, inhibition of the exocellular dd‐carboxypeptidase‐trans‐peptidase of Streptomyces R61 by β‐lactam antibiotics was competitive with regard to the donor substrate. However, the complexes formed between the Streptomyces R61 enzyme and various β‐lactam antibiotics were relatively stable, exhibiting half‐lives of 40 to 80 min at 37°C and neutral pH. During breakdown of the complexes the protein underwent reactivation, whereas the released antibiotic molecule was chemically altered. With [14C]benzylpenicillin, the released compound was neither benzylpenicillin nor benzylpenicilloic acid. The properties of the Streptomyces R61 enzyme β‐lactam antibiotic complexes were compared with those of the complexes formed between the same antibiotics and either the membrane‐bound transpeptidase from Streptomyces R61 or the exocellular dd‐carboxypeptidase‐transpeptidase of Streptomyces R39.
Structural Studies of Human‐Liver Alcohol‐Dehydrogenase Isoenzymes
Authors: BERGER Denis , BERGER Marianne , VON WARTBURG Jean‐Pierre
Published: March 2005

Alcohol dehydrogenase isoenzymes BE were isolated from human livers of both “normal” and “atypical” phenotypes [1]. Their pH‐rate profiles, their behaviour on CM‐cellulose and the number of hybrids formed with horse subunit A show that the “atypical” isoenzymes contain subunits B1 and B2 the “normal” one only subunit B1. These results indicate that most “atypical” individuals genetically represent heterozygotes.

A high degree of homology between horse subunit A and human subunits B is found. However, the human isoenzymes have a molecular weight of 87000 and contain about 410 amino acid residues, 6 tyrosines and 3 tryptophans per subunit.

The sequence of one tryptic peptide from subunit B1 is Phe‐Ala‐Lys and is altered to Phe‐Pro‐Lys in the subunit B2. This substituted residue is located in a region which corresponds to the coenzyme‐binding site of the horse enzyme.
Escherichia coli Ferredoxin, an Iron‐Sulfur Protein of the Adrenodoxin Type
Authors: KNOELL Hans‐Emil , KNAPPE Joachim
Published: March 2005

Ferredoxin of Escherichia coli is an iron‐sulfur protein of the molecular weight about 12000 which contains two iron and two labile sulfur atoms. The sequence of the amino terminus is HN‐Pro‐Lys‐Val‐Ile‐Val‐Leu‐Tyr‐.

The electron paramagnetic resonance spectrum of the reduced form shows a low‐field signal with g∥= 2.02 and a single high‐field signal with g⊥= 1.94. From the circular dichroism spectrum in the peptide absorption region an amount of 10% helically ordered regions is computed. A new peak in the light absorption spectrum at 545 nm appears upon reduction. From the spectral data, which reflect the fine structure of the chromophore and the gross conformation of the polypeptide chain, a close relationship to the mitochondrial adrenodoxin is concluded.

The midpoint potential of the protein is –0.38 V. It does not mediate electron transport in the NADP‐photoreduction system of spinach and is incapable of replacing the Pseudomonas putida ferredoxin in the camphor hydroxylation. The physiological role of this ferredoxin, which is not a functional analogue of the Escherichia coli flavodoxin and which is synthesized by aerobically as well as anaerobically growing cells, is unknown.
Isolation and Characterisation of Glycoproteins from Sputum
Authors: ROBERTS Glyn P.
Published: March 2005

Sputum has been separated into a sol and a gel phase by ultracentrifugation and the components of these phases examined. A glycoprotein of high molecular weight as well as serum albumin, immunoglobulin A, α1 acid glycoprotein and lactoferrin were detected in the sol phase by gel filtration and immunodiffusion studies. A glycoprotein of similar chemical composition was isolated from the gel phase by solubilisation in 6 M urea/0.1 M NaCl followed by fractional precipitation with ethanol. The carbohydrate content of this glycoprotein, termed the bronchial glycoprotein, varied from 58% (w/w) to 69% (w/w) with sputum from different patients and was composed of fucose, galactose, galactosamine, glucosamine and N‐acetylneuraminic acid. Serine, threonine and proline constituted 44% (w/w) of the amino acid residues of the bronchial glycoprotein and an alkaline borohydride cleavage study indicated that O‐glycosidic linkages exist between N‐acetylgalactosamine and the hydroxyamino acids in the peptide core. Fractionation of the bronchial glycoprotein on DEAE‐Sephadex A‐25 yielded fractions enriched in sulphate and sialic acid but separate fuco, sialo and sulpho glycoproteins were not obtained. Similarly fractionation of the oligosaccharides released by the action of alkaline borohydride yielded a complex mixture of oligosaccharides but sulphated oligosaccharides free of sialic acid were not obtained. Separation of the oligosaccharides into two fractions of different sizes showed that sulphate content was greatest in the larger oligosaccharides whereas sialic acid content was highest in the smaller oligosaccharides. A methylation study of the bronchial glycoprotein, neuraminidase‐digested bronchial glycoprotein and mild acid‐hydrolysed bronchial glycoprotein revealed that the N‐acetylneuraminic acid was linked to C‐3 of penultimate galactose units. Fucose occurs in three different linkages, approximately 50% is linked to C‐2 of penultimate galactose units, approximately 25% is linked to C‐4 of glucosamine residues already substituted at C‐3 and the remainder is linked to C‐3 of glucosamine residues already substituted at C‐4.
Induction of Polyadenylate Polymerase and Differentiation in Neuroblastoma Cells
Authors: SIMANTOV Rabi , SACHS Leo
Published: March 2005

Treatment of neuroblastoma cells with dibutyryl‐adenosine 3′:5′‐monophosphate or adenine induced axon formation and a three‐fold increase in the polyadenylate, poly(A), content of the polysomal mRNA. The extracted poly(A) contained 90% adenylic acid and showed a mobility of 6–7 S in dodecylsulfate‐polyacrylamide gel electrophoresis. Treatment with dibutyryl‐adenosine 3′:5′‐monophosphate or adenine, also induced a 4–6‐fold increase in a nuclear enzymic activity that incorporated [3H]ATP to an acid‐insoluble polymer in a cell‐free system. This polymer, like poly(A) extracted from the polysomal mRNA, was bound at high salt concentration to nitrocellulose filters. [3H]ATP incorporation was Mg2+‐dependent, sensitive to ribonuclease and EDTA and resistant to deoxyribonuclease and actinomycin D. There was no incorporation of [3H]UTP or [3H]dTTP and addition of UTP, CTP and GTP did not increase the incorporation of [3H]ATP. 5‐Bromodeoxyuridine induced axon formation of neuroblastoma cells and poly(A) polymerase activity, without increasing the poly(A) content in the polysomal mRNA. The results indicate that induction of axon formation of neuroblastoma cells is associated with an increase in the activity of poly(A) polymerase. It is suggested that the induction of this enzyme may be generally involved in cell differentiation.
Rat‐Liver Cholesterol 7α‐Hydroxylase
Authors: GIELEN Jacques , VAN CANTFORT Jacques , ROBAYE Bernadette , RENSON Jean
Published: March 2005

The establishement of the circadian rhythm of cholesterol 7α‐hydroxylase activity requires protein and RNA synthesis. The spontaneous decrease of the enzymic activity, at the end of the night, allows us to evaluate a half‐life time of about two hours. The half‐life time goes up to about four hours when the enzymatic activity decay is measured following cycloheximide administration. This difference suggests that an active mechanism is involved in the control of the enzyme degradation. The daily variation of the enzyme activity is regulated via the hypothalamo‐hypophysis‐adrenal axis. At the cellular level glucocorticoids are the most likely responsible agent. The hepatic cholesterol 7α‐hydroxylase variations always parallel the plasmatic corticosterone concentration fluctuations, the latter being by far the most important adrenocortical excretion product. These two rhythms are modified in a similar manner under different physio‐pathological conditions, such as the inversion of lighting in the animal room or the inversion of feeding time. Of these two parameters, the moment of food intake is the most important and accounts for the synchronisation of the rhythm in the animals. The rhythm is retained after several days of starvation but its amplitude decreases and the individual variations among the animals increase significantly at each time point.
The Active Species of ‘CO2’ Utilized by Reduced Ferredoxin: CO2 Oxidoreductase from Clostridium pasteurianum
Authors: THAUER Rudolf K. , KÄUFER Barbara , FUCHS Georg
Published: March 2005

Reduced ferredoxin: CO2 oxidoreductase (CO2 reductase) from Clostridium pasteurianum catalyzes the reduction of ‘CO2’ to formate with reduced ferredoxin, an isotopic exchange between ‘CO2’ and formate in the absence of ferredoxin, and the oxidation of formate to ‘CO2’ with oxidized ferredoxin. The active species of ‘CO2’, i.e. CO2 or HCO3− (H2CO3), utilized by the enzyme was determined. The method employed for the species identification was that of Cooper et al. (1968). Both ‘CO2’ reduction to formate and the exchange reaction were studied. Data were obtained which are compatible with those expected if CO2 is the active species.

The V and the dissociation constant Ks of the enzyme · CO2 complex in dependence of pH were determined from initial velocity studies of the exchange reaction. V was found to be only slightly affected by pH between 5.5 and 7.5. Ks was markedly dependent on pH; the constant increased with decreasing pH from 0.2 mM at pH 7.5 to 3 mM at pH 5.5.
Initiation of Transcription within an RNA‐Polymerase Binding Site
Authors: HEYDEN Bertold , NÜSSLEIN Christiane , SCHALLER Heinz
Published: March 2005

The 5′‐terminal sequence of the RNA transcribed from bacteriophage fd replicative form DNA under the control of promoter region I has been determined to be ppp(Gp)nUpApApApGpApCpCpUpGpApUpUp …

This sequence is complementary to the 5′‐terminal sequence of the minus strand of the corresponding RNA polymerase binding site I, the starting point for RNA synthesis lying approximately in the middle of the binding site.

This initial sequence is also transcribed faithfully from isolated complexes of RNA polymerase and binding site I, obtained by DNase digestion of complexes between RNA polymerase and fd replicative form DNA. These highly stable complexes can not be reconstituted from binding site and enzyme.

It is concluded that RNA polymerase binding site and initiation site are identical parts of a promoter region, and that no “drift” between these sites is required as a step in RNA chain initiation. An additional non‐transcribed outside region is implicated as essential for full promoter function.
Association of Glyceraldehyde‐3‐Phosphate Dehydrogenase with the Particulate Fraction of Chicken Skeletal Muscle
Authors: DAGHER Shawky M. , HULTIN Herbert O.
Published: March 2005

When chicken breast muscle was homogenized in water, approximately 86% of the glyceraldehyde‐3‐phosphate dehydrogenase was associated with the particulate fraction.

The enzyme was solubilized by increasing pH with a very marked increase in the pH range of 6.9 to 7.1. At low ionic strength (about 0.015), approximately 50% of the enzyme is solubilized at pH 7.5 and above.

Increasing ionic strength also led to increased solubilization. In addition, there was a specific cation effect with Ca2+ > Mg2+ > K+ > Na+ at a constant ionic strength. Glyceraldehyde 3‐phosphate and 2,3‐bisphosphoglycerate were effective in partially solubilizing the enzyme.

Solubilized glyceraldehyde‐3‐phosphate dehydrogenase can rebind to the particulate fraction of the homogenized muscle. The soluble form of the enzyme has a higher V and a higher Km (glyceraldehyde 3‐phosphate) than the enzyme bound to the particulate fraction.
Affinity Chromatography on Immobilised Nucleotides
Authors: COMER Michael J. , ATKINSON Anthony , CRAVEN David B. , HARVEY Michael J. , DEAN Peter D. G.
Published: March 2005

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose has been examined. Specific elution from the substituted AMP‐Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP‐Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant.

The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde‐3‐phosphate dehydrogenase to 6‐aminohexanoyl‐NAD+‐Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde‐3‐phosphate dehydrogenase from partially purified extracts of this organism was achieved.
Broad‐Specificity Proteinase Inhibitors in Scopolia japonica (Solanaceae) Cultured Cells
Authors: SAKATO Kuniaki , TANAKA Hozumi , MISAWA Masanaru
Published: March 2005

Proteinase inhibitors were isolated from Scopolia japonica cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP‐20, decolorization by Duolite A‐7, affinity chromatography on trypsin‐Sepharose, and Bio‐Gel P‐4 chromatography. It was found that the proteinase inhibitors from S. japonica cells are a mixture of at least five components.

For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15%α‐helical conformation by the Moffitt‐Yang analysis of ORD data.

The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non‐competitive type.
Changes in the State of Actin during Superprecipitation of Actomyosin
Authors: STRZELECKA‐GOLASZEWSKA Hanna , DRABIKOWSKI Witold , JAKUBIAK Miroslawa
Published: March 2005

Exchangeability of actin‐bound ADP and calcium in actomyosin at low ionic strength has been studied using F‐actin labelled with [14C]ADP or 45Ca and measuring release of radioactivity into solution. Low‐speed centrifugation, ultracentrifugation and ultrafiltration were used to separate protein from the medium. Comparison of the results obtained with these three separation procedures has revealed that the release of [14C]ADP and 45Ca into the medium in the presence of millimolar concentrations of MgATP is largely due to the release under these conditions of actin itself retaining its bound ADP and calcium. The real exchange of the bound nucleotide and calcium, even under the most favourable conditions, was in our experiments limited to about 20%. Detailed examination of the dependence of both the release of actin and the exchange of actin‐bound ADP and calcium on the free divalent cations present, the kind and concentration of the added nucleotide, and temperature of incubation indicates that there is no correlation between the exchange and superprecipitation of actomyosin. The results presented support the view that the limited enhancement by myosin of the exchange of nucleotide and cation bound to actin under certain conditions results from accidental disruption of bonds between actin monomers due to a mechanical stress exerted on actin filaments upon their interaction with myosin filaments.