bluuebunny/crossref_metadata_2025 huggingface dataset:
The Purification and Characterisation of the Human‐Serum Binding Protein for the 25‐Hydroxycholecalciferol (Transcalciferin) Identity with Group‐Specific Component
Authors: BOUILLON Roger , VAN BAELEN Hugo , ROMBAUTS Wilfried , DE MOOR Pieter
Published: March 2005

The binding protein for 25‐hydroxyvitamin D3 has been isolated from human serum by monitoring the recovery of 3H‐labeled 25‐hydroxyvitamin D3. After a 500‐fold purification a pure protein was obtained as judged from the constant specific activity (ratio of absorbance versus radioactivity) on agarose and DEAE‐Sephadex chromatography and on the presence of a single band on both cellulose acetate and polyacrylamide gel electrophoresis.

The molecular weight of the purified protein was measured by gel filtration on agarose (56000), Sephadex G‐75 (58000) and dodecylsulfate‐polyacrylamide gel electrophoresis (56000). On sucrose gradient ultracentrifugation a sedimentation coefficient of 4.1 S was found. The isoelectric point was 4.89 S on isoelectric focusing. The stability of the protein at 60°C was enhanced by the presence of excess 25‐hydroxyvitamin D3. On tandem crossed immunoelectrophoresis the purified binding protein was found to be identical to the one present in whole serum.

The activity of the isolated protein was demonstrated by a Ka at 4 C of 1.2 1010 I mol−1. A binding capacity of 0.8 binding site/molecule was measured on a Sephadex G‐25 column.

During immunological studies with this protein it became evident that the binding protein is identical with another serum protein known as group‐specific component (Gc).

In analogy to other serum binding proteins we propose to call this group‐specific component/25‐hydroxyvitamin‐D3‐binding protein transcalciferin.
On the Kinetics of the Rifampicin RNA‐Polymerase Complex Differences between Crude and Purified Enzyme Fractions
Authors: HANDSCHIN Jürg C. , WEHRLI Walter
Published: March 2005

The antibiotic rifampicin forms a very tight complex with DNA‐dependent RNA polymerase of Escherichia coli. The rate constants of association and dissociation of this complex have been measured and found to be dependent on the purity of the enzyme. Thus a crude RNA polymerase (fraction‐3 enzyme) has rate constants different from those of an enzyme further purified by DEAE‐cellulose chromatography (fraction‐4 enzyme). The complex produced by the antibiotic and the fraction‐3 enzyme is about ten times more stable and is formed about ten times more slowly than the complex with fraction‐4 enzyme. It has been shown that the RNA present in the crude enzyme and removed by chromatography on DEAE‐cellulose is the cause of the change in the kinetics of the complex. tRNA of rat liver and crude rat liver RNA added to purified RNA polymerase have a similar effect. Mg2+, which has no intrinsic influence, augments the effect of the nucleic acids, whereas monovalent cations do not. Since nucleic acids increase the stability of the complex, but at the same time decrease the rate of its formation, the equilibrium constant, Keq, remains almost the same. The possible effects of nucleic acids on the rifampicin binding site are discussed.
Immunochemical Isolation and Characterization of Vitellogenin mRNA from Liver of Estradiol‐Treated Chicks
Authors: JOST Jean‐Pierre , PEHLING Gundula
Published: March 2005

Vitellogenin mRNA was purified through three steps. A heavy polysome fraction was obtained by discontinuous sucrose density gradient centrifugation, vitellogenin polysomes were immunoprecipitated with affinity‐chromatography‐purified anti‐lipovitellin IgG and goat anti‐rabbit IgG, the enriched mRNA was isolated on a poly(U)‐Sepharose column. As judged by its specific activity in a reticulocyte lysate system, vitellogenin mRNA has been enriched a 1000‐fold with a recovery of 30%On 99% formamide 3.4 % polyacrylamide gels vitellogenin mRNA has an Mr of 2.4–2.5 × 106 and codes for a peptide of Mr 240000, which under our incubation conditions is partially degraded to smaller peptides.
Carboxymethylated Liver Alcohol Dehydrogenase
Authors: HAREMAN Michael J.
Published: March 2005

The rate constants for NADH binding and dissociation for carboxymethylated alcohol dehydrogenase have been determined and compared to those for the native enzyme.

Steady‐state and transient kinetic experiments Lave shown that the hydrogen transfer step is rate‐determining for oxidation of ethanol by carboxymethylated alcohol dehydrogenase. The rate constant of 0.19 s−1 is considerably slower than that for the native enzyme.

The steady‐state parameter, V/[E], was obtained for each of a series of alcohols and correlated with the Taft σ* parameter. The linear relationship obtained indicates that the same step, hydrogen transfer, is rate‐determining for all the alcohols. The Q* value obtained is the same as for the native enzyme; the implications of this for the mechanism of hydrogen transfer are discussed.
Studies on the Structure and Function of Chick‐Oviduct Chromatin
Authors: STRÄTLING Wolf H. , O'MALLEY Bert W.
Published: March 2005

Chromatin prepared at various stages of hormone‐mediated development of the chick oviduct was investigated for the relative proportions of transcriptionally active (fraction I) and repressed (fraction II) fractions by ECTHAM‐cellulose chromatography. During primary stimulation with estrogen, the amount of chromatin DNA in fraction I plotted as a function of time of stimulation showed a bell‐shaped profile, similar to the profile obtained earlier for the number of chromatin sites available to RNA polymerase for initiation of RNA synthesis. Chromatin from a transcriptionally inactive system, hen erythrocytes, eluted mainly (98 %) as fraction II.

The, transcriptionally active fraction I of estrogen‐stimulated oviduct contained a 4‐fold greater RNA polymerase II activity than was found in fraction II. This could be explained by a differential inhibition of RNA polymerase activity in fraction II since enzyme preparations extracted and purified from both chromatin fractions showed equal activities. In support of this finding, fraction I eluted from ECTHAM‐cellulose showed a 4‐fold greater concentration of rifampicin‐resistant RNA chain initiation sites as compared to fraction II.

When chromatin from oviduct mince was incubated with labeled progesterone and 17β‐estradiol and was chromafographed on ECTHAM‐cellulose, the transcriptionally active fraction also contained a 4‐fold greater concentration of bound hormone (per weight DNA) as compared to the repressed fraction
Size, Complexity and Abundance of a Specific Poly(A)‐Containing RNA of Liver from Male Xenopus Induced to Vitellogenin Synthesis by Estrogen
Authors: WAHLI Walter , WYLER Toni , WEBER Rudolf , RYFFEL Gerhart U.
Published: March 2005

Estrogen treatment of Xenopus males leads to the appearance of a new species of poly(A)‐containing RNA in the liver, at a stage when large amounts of the estrogen‐induced yolk precursor protein, vitellogenin, is produced. This estrogen‐induced RNA sediments at 28 S and migrates on gels in aqueous solution with an apparent molecular weight of 2.0 × 106. Contour length measurements under denatiuring conditions in the electron microscope reveal a molecular weight of 2.34 × 106 compared to the mouse 28‐S rRNA.

Labeling experiments show that the estrogen‐induced RNA has a higher stability than the average liver poly(A)‐containing RNA and represents 10–20% of the poly(A)‐containing RNA in the cytoplasm after 24 h of labeling.

Hybridization of complementary DNA, synthesized on the. isolated estrogen‐induced RNA, with its template suggests a complexity corresponding to a single species of poly(A)‐containing RNA of such a high molecular weight. Hybridization of the complementary DNA with cytoplasmic poly(A)‐containing RNA from estrogen‐treated Xenopus males and control toads show that the estrogen‐induced RNA constitutes 12–15% of all cytoplasmic poly(A)‐containing RNA, and is at least 2000‐fold less abundant in untreated males.

Size, complexity and abundance of the estrogen‐induced RNA are characteristics expected for a mRNA coding for vitellogenin.
Affinity Chromatography and Binding Studies on Immobilized Adenosine 5′‐Monophosphate and Adenosine 2′,5′‐Bisphosphateof Nicotinamide Nucleotide Transhydrogenasefrom Pseudomonas aeruginosa
Authors: HÖJEBERG Bo , BRODELIUS Peter , RYDSTRÖM Jan , MOSBACH Klaus
Published: March 2005

Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6‐(6‐aminoyl)‐adenosine‐2′,5′‐bisphosphate‐Sepharose 4B.

Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 ± 2000.

The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2′‐monophosphate and Ca2+were activators whereas NADP+ was inhibitory.

Nicotinamide nucleotide‐specific release of binding of the transhydrogenase to N6‐(6‐aminohexyl)‐adenosine‐2′,5′‐bisphosphate‐Sepharose and N6‐(6‐aminohexyl)‐adenosine‐5′‐monophospliate‐Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H).

Binding of transhydrogenase to N6‐(6‐aminohexyl)‐adenosine‐2′,5′‐bisphosphate‐Sepharose and activation of the enzyme by adenosine‐2′,5′‐bisphosphate showed a marked pH dependence. In contrast, inhibition of the Ca2+‐activated enzyme by adenosine 2′,5′‐bisphosphate was virtually constant at various pH values. This discrepancy was interpreted to indicate the existence of separate nucleotide‐binding effector and active sites.
Phosphorylation of Intracellular Fructose in Bacillus subtilis Mediated by Phosphoenolpyruvate‐l‐Fructose Phosphotransferase
Authors: DELOBBE André , CHALUMEAU Hervé , CLAVERIE Jean‐Michel , GAY Philippe
Published: March 2005

Intracellular fructose provided by the sorbitol pathway in Bacillus subtilis, can be phosphorylated by the phosphoenolpyruvate‐1‐fructose phosphotransferase which is known to mediate a vectorial metabolism. The fate of this intracellular fructose was studied using mutants lacking either the fructose 1‐phosphate pathway or the fructose 6‐phosphate pathway.

It was shown that the phosphoenolpyruvate‐dependent phosphorylation needs a prior exit of the sugar into the medium, this exit being probably catalysed by a transport system. A low affinity intracellular phosphoenolpyruvate phosphotransferase system was found, which seems to be devoid of a physiological role.
Spatial Characteristics of the Aspartate Membrane Carrier and Its Relationship to the Glutamate Transport System in Membrane Vesicles of Escherichia coli K‐12 Cavalli
Authors: KAHANE Simona , METZER Esther , HALPERN Yeheskel S.
Published: March 2005

Membrane vesicles of Escherichia coli K‐12 CS7, a strain genetically derepressed for glutamate permease, maintain low aspartate transport activity, like that of preparations of the wild‐type parent. Growth of the parent CS101 on aspartate as the source of carbon or nitrogen results in derepression of both aspartate and glutamate transport. Growth of strain CS7 on aspartate derepresses aspartate transport to the same extent as in strain CS101, but only slightly increases the derepressed level of glutamate transport activity.

The affinity of the membrane transport system for glutamate is enhanced by sodium, while that for aspartate is not.

Although the affinities for glutamate (23 μM) and aspartate (12 μM) are similar, aspartate does not inhibit glutamate transport, while glutamate competitively inhibits aspartate transport.

Aspartate transport, but not glutamate transport, is competitively inhibited by C4 dicarboxylic acids, whereas 2‐oxoglutarate competitively inhibits glutamate transport, but not aspartate transport.

Competitive inhibition of l‐aspartate transport by l‐glutamate and by the 5‐methyl ester of l‐glutamate is abolished in the presence of 2‐oxoglutarate. However, 2‐oxoglutarate does not affect the competitive inhibition of l‐aspartate transport by d‐aspartate and by dl‐threo‐3‐hydroxyaspartate. The relationship between the two dicarboxylic amino acid transport systems and the spatial characteristics of the aspartate carrier are discussed in the light of these findings.
Paromomycin and Dihydrostreptomycin Binding to Escherichia coli Ribosomes
Authors: LANDO Danielle , COUSIN Marie‐Anne , OJASOO Tiiu , RAYNAUD Jean‐Pierre
Published: March 2005

Paromomycin binds specifically to a single type of binding site on the 70‐S streptomycin‐sensitive Escherichia coli ribosome. This site is different from that of dihydrostreptomycin since paromomycin binds to streptomycin‐resistant ribosomes and since dihydrostreptomycin does not compete for paromomycin binding. Paromomycin binding, unlike dihydrostreptomycin binding, is independent of changes in ribosome concentration but influenced by magnesium ion concentration. Moreover, paromomycin does not bind to the 30‐S subunit of the streptomycin‐sensitive ribosome, except in the presence of dihydrostreptomycin, which probably induces the conformational changes necessary for a paromomycin binding site. This induction does not occur with streptomycin‐resistant ribosomes. Neither antibiotic binds to the 50‐S subunit. In general, binding of the one antibiotic increases the number of sites available for binding of the other. Both antibiotics hibit marked non‐specific binding at high antibiotic/ribosome ratios. Competition studies have enabled the classification of other aminoglycosides according to their ability to compete for the paromomycin and dihydrostreptomycin binding sites. Derivatives structurally related to paromomycin compete for its binding, the degree of competition being related to antibacterial activity, but do not compete for dihydrostreptomycin binding; they, on the contrary, increase the number of dihydrostreptomycin binding sites. Neither gentamicin nor kanamycin derivatives, which induce a high level of misreading, nor kasugamycin and spectinomycin, which do not induce misreading, compete for paromomycin or dihydrostreptomycin binding sites. Other sites may be involved in the binding of these aminoglycosides and in inducing misreading.
Substituent Effect on the Oxidation of Phenols and Aromatic Amines by Horseradish Peroxidase Compound I
Authors: JOB Dominique , DUNFORD H. Brian
Published: March 2005

A stopped‐flow kinetic study shows that the reduction rate of horseradish peroxidase compound I by phenols and aromatic amines is greatly dependent upon the substituent effect on the benzene ring. Moreover it has been possible to relate the reduction rate constants with the ionization constants of monosubstitutcd substrates by a linear free‐energy relationship (Hammett equation). The correlation of log (rate constants) with σ values (Hammett equation) and the absence of correlation with σ+ values (Okatnoto‐Brown equation) can be explained by a mechanism of aromatic substrate oxidations, in which the substrate gives an electron to the enzyme compound I and simultaneously loses a proton. The analogy which has been made with oxidation potentials of phenols or anilines strengthens the view that the reaction is only dependent on the relative ease of oxidation of the substrate. The rate constant obtained for p‐aminophenol indicates that a value of 2.3 × 108 M−1 s−1probably approaches the diffusion‐controlled limit for a bimolecular reaction involving compound I and an aromatic substrate.
The Role of Escherichia coli dnaA Gene and Its Integrative Suppression in M 13 Coliphage DNA Synthesis
Authors: MITRA Sankar , STALLIONS Donald R.
Published: March 2005

An F+ derivative of Escherichia coli E 508 thermosensitive in dnaA function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E 508(ts) in which the temperature‐sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M 13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature‐sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature‐sensitive mutant also, but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dnaA function: (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature‐sensitive dnaA mutant at the nonpermissive temperature; (c) progeny single‐strand DNA synthesis does not absolutely require dnaA function; (d) progeny single‐strand DNA is present in the circular form. The implication of the host DNA replication in M 13 DNA synthesis is discussed.
Resolution and Partial Characterization of a Low‐Molecular‐Weight Product of Protein Synthesis in Isolated Rat‐Liver Mitochondria
Authors: DIANOUX Anne‐Christine , BOF Mireille , CÉSARINI René , REBOUL Angeline , VIGNAIS Pierre V.
Published: March 2005

A low molecular weight product (Mr 7000) synthetized on rat liver mitoribosomes is selectively extracted by chloroform/methanol (2/1). This product is characterized by an unusual hydrophobicity as reflected by its chromatographic behaviour, its high content in hydrophobic amino acids and its strong capacity to bind lipids. Bound lipids contain a high percentage of linoleic acid.
Nicotinamide‐Dependent One‐Electron and Two‐Electron (Flavin) Oxidoreduction : Thermodynamics, Kinetics, and Mechanism
Authors: BLANKENHORN Gunter
Published: March 2005

Biological nicotinamide‐dependent oxidoreduction consists of reversible 2e‐oxidoreduction of substrates. A mechanism involving subsequent le‐steps is shown to be very unfavourable due to the high energy of the nicotinamide radical.

Free energy relationships provide a convenient tool, allowing one to differentiate between hydride transfer and hydrogen atom transfer. It is concluded that biological nicotinamide‐dependent, as well as flavin‐nicotinamide oxidoreduction, proceed via hydride transfer but not via hydrogen atom transfer.

In flavin‐nicotinamide oxidoreduction, flavin‐nicotinamide charge transfer complexes are very likely the catalytic intermediates, preceding transfer of hydride ion. The energy of the long‐wavelength charge transfer transition of zwitterionic oxidized‐nicotinamide/reduced‐flavin complexes is strongly dependent on polarity. It is maximal in a highly polar environment.

5‐Deazaflavins show the high thermodynamic radical instability of nicotinamides. They have to be considered as nicotinamide analog 2e‐oxidoreductants rather than flavin analogs, therefore, lacking the ability to catalyze reversible le‐oxidoreduction, essential for many flavoenzymes.
The Energetics of Escherichia coli during Aerobic Growth in Continuous Culture
Authors: FARMER Ian S. , JONES Colin W.
Published: March 2005

The energetics of Escherichia coli W growing aerobically in continuous culture have been investigated. Conditions were chosen such that growth was limited by the availability of carbon or oxygen (energy‐limited cultures), or of ammonium or sulphate ions (excess energy cultures).

Under glycerol‐limited conditions YmaxO2(true molar growth yield with respect to oxygen) and YmaxATP (true molar growth yield with respect to ATP equivalents) were 50.9 g cells · mol O2−1 and 12.7 g cells mol ATP equivalents−1 respectively; these values were not substantially altered during growth limited by oxygen. ammonium or sulphate. In contrast, M (the energy requirement. for maintenance purposes) increased from approximately 2 mmol ATP equivalents · h−1 g.cell−1 during energy‐limited growth to 16.8 and 30.8 mmole ATP equivalents h−1 g cells−1 when growth was limited by ammonium and sulphate ions respectively.

Replacement of glycerol by other limiting carbon sources caused YmaxATP to alter within the range 13.9 (glucose) to 7.1 (acetate) g cells mol ATP equivalents−1 in the order glucose > galactose > arabinose > fructose > glycerol > fumarate > lactate > pyruvate > acetate. In each case the experimental value of YmaxATP was ≤ 55% of the theoretical value calculated from the known energy requirements for the biosynthesis of cell materials.

It is concluded from these results that neither M nor YmaxATP are constant values for E. coli. M varies with the energy supply, being highest under excess energy growth conditions where it may reflect energy wastage by the cell. On the other hand, YmaxATP varies with the nature of the growth substrate and thus reflects the different energy requirements for the synthesis of cell material from different carbon sources
An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates
Authors: PELHAM Hugh R. B. , JACKSON Richard J.
Published: March 2005

A simple method is described for converting a standard rabbit reticulocyte cell‐free extract (lysate) into an mRNA‐dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol‐bis(2‐aminoethylether)‐N,N′‐tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full‐sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.
Structure‐Activity Relationships in Acetylcholinesterase Reactions
Authors: JÄRV Jaak , KESVATERA Tônu , AAVIKSAAR Aavo
Published: March 2005

The Michaelis‐Menten parameters kcat Ks(app) and the second‐order rate constants k11= k2/Ks of acetylcholinesterase‐catalyzed hydrolysis of 25 acetic esters with note‐ionic leaving groups have been determined at 25 °C and pH 7.5 in 0.15 M KCL A linear relationship between the substrate non‐covalent binding capacity and the leaving group hydrophobicity, and a multiparameter correlation of the acetylation reaction rate constant logarithm with the leaving group, inductive effect, hydrophobicity, and steric effect, have been established. The acetyl‐enzyme deacetylation rate constant has been calculated. Taken together, a fairly complete understanding of acetylcholinesterase specificity is possible.

The data are consistent with a model of the acetylcholinesterase active site, in which the catalytically active groups are located at the bottom of a jaws‐like slit with a limited range of hydrophobic walls that provide the sorption of the substrate leaving groups not longer than that in n‐butyl acetate.
The Transfer of J Blood‐Group Activity from J‐Containing Bovine Serum to J‐Negative Erythrocytes
Authors: KRÖTLINGER Franc , THIELE Otto Wolfgang , OHL Cornelia
Published: March 2005

The J blood‐group activity of bovine serum is contained both in a lipid and in a nonlipid fraction. This is also true for calf serum.

It is demonstrated that the J determinant is transferred from a serum protein onto the erythrocyte membrane by incubation in vitro. Even though the donor of J activity is a lipid‐free serum protein (probably a glycoprotein), the transferred J activity is detectable only in the lipid fraction of erythrocytes. Thus, the J determinant (probably a carbohydrate unit) must have been detached from a serum glycoprotein and transferred to a lipidic receptor (probably a glycosphingolipid) at the erythrocyte membrane.

It is suggested that an enzyme system located in or at the erythrocyte membrane is responsible for the transfer of J substance.

The transfer of J substance is inhibited by a polar lipid present in bovine serum.
Isolation and Characterization of a Growth‐Cycle‐Reflecting, High‐Molecular‐Weight Protein Associated with Escherichia coli Ribosomes
Authors: SUBRAMANIAN Alap R. , HAASE Cordula , GIESEN Marcella
Published: March 2005

A high‐molecular‐weight, acidic protein whose amount per ribosomal particle depends on the growth cycle is shown to be associated with Escherichia coli ribosomes. The protein is associated with ribosomes from cells harvested at different phases of the growth cycle, but a large increase in its amount is seen with ribosomes from post‐exponential phase cells. The protein is only slightly washed off the ribosomes by 1 M NH4Cl. When ribosomes cue dissociated it remains entirely with the 30‐S subunits. We have purified the protein to homogeneity. It has a molecular weight of 70000 and its amino acid composition showed some resemblance to that of ribosomal protein S1. However, the two proteins are shown to be different. These results, together with the earlier‐shown variation in the acetylation of L12 during the growth cycle, indicate that certain proteins of (or associated with) E. coli ribosomes may carry specific biochemical roles connected with cellular adaption toward the stationary phase.
Specificity and Properties of the Destabilization, Induced by Initiation Factor IF‐3, of Ternary Complexes of the 30‐S Ribosomal Subunit, Aminoacyl‐tRNA and Polynucleotides
Authors: RISULEO Gianfranco , GUALERZI Claudio , PON Cynthia
Published: March 2005

Initiation factor IF‐3 causes the destabilization of preformed ternary complexes of 30‐S ribosomal subunit, codons and aminoacyl‐tRNAs or peptidyl‐tRNA. This destabilization is dilution‐dependent and affects all ternary complexes with the exception of those containing the initiator fMet‐tRNA, which remain more resistant to IF‐3‐induced destabilization under the various conditions studied. Several possible reasons for this specificity have been examined. It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing fMet‐tRNA, (b) the amount of aminoacyl‐tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group. On the other hand, the nature of the polynucleotide in response to which the, ternary complex is formed was found to influence the amount of aminoacyl‐tRNA bound to the ribosome, and to some extent the amount of aminoacyl‐tRNA which can be released. The ternary complex containing the mischarged initiator tRNA fVal‐tRNAfMet displays greater resistance to the IF‐3‐induced destabilization than the complex containing fVal‐tRNAVal. These results indicate that the specificity of the IF‐3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide. The IF‐3‐induced destabilization of ternary complexes was found to be little affected by variations in reaction conditions, so that this IF‐3 activity can be used to measure the stoichiometric binding of IF‐3 to the ribosome over a broad range of pH and K+ and Mg2+ concentrations. Several antibiotics have been tested for their capacity to interfere with this reaction; only high concentrations of tetracycline blocked this IF‐3 activity.
Diglyceride Kinase Activity of Microtubules
Authors: DALEO Gustavo R. , PIRAS Marta M. , PIRAS Romano
Published: March 2005

Vinblastine‐isolated microtubule protein from chick embryonic muscles has an enzymatic activity which catalyzes the formation of phosphatidic acid from diglycerides and ATP. The pH optimum (6.4), sedimentation on sucrose gradients (Mr= 85000), and sensitivity to ions of this diglyceride kinase activity are different to those of a similar enzymatic activity present in 150000xg supernatants of chick embryonic muscle homogenates, suggesting that it is a different species which is associated specifically with the microtubules. The reaction requires a divalent ion (e.g. 0.4 mM Mg2+ gives half‐maximal stimulation), and GTP can replace ATP rather effectively, especially at nucleotide concentrations lower than 50 μM. The sedimentation of the diglyceride kinase on sucrose gradients coincides with that of the microtubules‐associated protein kinase (Mr= 75000): the heat‐stability and sensitivity to proteolysis of both activities are also very similar. Stimulation of one reaction by the addition of the corresponding exogenous substrate does not impair the phosphorylation of the other, and no radioactivity is lost from phosphatidic acid or the protein moiety upon incubation of pre‐labelled microtubules with a large excess of unlabelled ATP or GTP.

In addition to diglyceride kinase and protein kinase activities (0.2 and 0.3 nmol 32P‐transferred × min−1× mg−1 microtubular protein, respectively), microtubules also contain an associated ATPase (2.8 nmol × min−1× mg−1), which requires either Mg2+ or Ca2+, can hydrolyze GTP quite effectively, and sediments with a molecular weight of 95000.

The results obtained are discussed in connection with the possible relationships existing among these enzymatic activities, as well as their probable role in microtubular functions.
Initiation of the Polypeptide Chain by Reticulocyte Cell‐Free Systems
Authors: FRESNO Manuel , CARRASCO Luis , VAZQUEZ David
Published: March 2005

In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met‐tRNAf IF‐E2 GTP, (b) binding of the initiator Met‐tRNAf to the 40‐S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) formation of the initiation complex with 80‐S ribosomes and (d) formation of the first peptide bond.

Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and showdomycin block the formation of the ternary complex Met‐tRNAf· IF‐E2 GTP. Edeine A1, aurintricarboxylic acid and polydextran sulphate block the binding of the mRNA to the 40‐S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met‐tRNAf to the 80‐S ribosome in the presence of initiation factors is observed with sparsomycin and antibiotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met‐tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin.

We have developed a simple technique to detect the Met‐tRNAf· 40‐S‐subunit poly(A,G,U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the formation of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described.
Role of GTP in Eukaryotic Polypeptide‐Chain Initiation
Authors: OCHIAI‐YANAGI Sonoe , MAZUMDER Rajarshi
Published: March 2005

A factor, which makes a ternary complex with GTP and eukaryotic initiator tRNA (Met‐ tRNAi), has been purified 100‐fold from developed cysts of Artemia salina. Some of the properties of the purified factor have been studied.

Mg2+ appears to inhibit ternary complex formation.

Little or no ternary complex is formed when 5 μ GTP is replaced by an identical concentration of UTP, CTP or ATP. The analog, guanosine 5′‐(β,γ‐imino)triphosphate [GMP‐P(NH)P] seems to be a much better substitute for GTP than guanosine 5′‐(β,γ‐methylene)triphosphate [GMP‐ P(CH2)P]. Since GMP‐P(NH)P is as effective as GTP in ternary complex formation, it would appear that GTP plays the role of an allosteric effector in this step of eukaryotic polypeptide chain initiation.

GDP inhibits both the rate and extent of ternary complex formation. The inhibition is largely reversed by adding a 5‐fold molar excess of GTP over GDP. dGDP is slightly less inhibitory than GDP. UDP and CDP are much less inhibitory than GDP and very little inhibition is obtained with ADP.

The preformed ternary complex is rapidly and completely destroyed in the presence of N‐ethylmaleimide. The results suggest that free –SH groups of the factor may be essential for maintaining the integrity of the ternary complex.
Effects of Thyroid Hormone Deficiency on the Distribution of Hepatic Metabolites and Control of Pathways of Carbohydrate Metabolism in Liver and Adipose Tissue of the Rat
Authors: BAQUER Najma Zaheer , CASCALES Maria , McLEAN Patricia , GREENBAUM A. Leslie
Published: March 2005

Measurements have been made of the hepatic metabolites in normal and thyroidectomized rats and of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and of lipogenesis together with the flux of glucose through alternative pathways of glucose metabolism and into lipid.

There is a significant fall in the content of ADP, AMP, citrate, long‐chain acyl‐CoA derivatives and a rise of 3‐phosphoglycerate, 2‐phosphoglycerate and phosphoenolpyruvate following thyroidectomy. The observed changes in the glycolytic intermediates may be correlated with the increased activity of phosphofructokinase relative to pyruvate kinase and fructose bisphosphatase.

The NAD+/NADH quotient of the mitochondrial compartment, calculated from the reactants and Keq of 3‐hydroxybutyrate dehydrogenase, becomes significantly more oxidized in hypothyroid animals. The redox state of the cytosolic NAD and NADP couples remains relatively unchanged.

The changes in the hepatic content of CoA derivatives and citrate and in the mitochondrial redox state are interpreted as indicating a depressed rate of lipid oxidation.

Calculations of the compartmentation of metabolites between the cytosol and mitochondria indicate a very marked decrease in mitochondrial citrate, 2‐oxoglutarate and glutamate with smaller changes in aspartate and malate. These changes are interpreted as providing evidence for the importance of modifications in the malate‐aspartate shuttle in hypothyroidism; this is further supported by measured changes in the distribution and activities of the component enzymes of the hydrogen shuttles.

There is a diminished activity of glucokinase and of enzymes of the glycolytic pathway below phosphofructokinase in livers from hypothyroid rats. The oxidative enzymes of the pentose phosphate pathway, ATP‐citrate lyase, ‘malic’ enzyme and fatty acid synthetase also decrease markedly. There is a striking parallelism between the changes of enzyme profile of liver and adipose tissue in hypothyroidism.

Adrenal glands from hypothyroid rats showed a generalized decline in enzyme activity in parallel with the fall in tissue weight. Cytosolic glycerol‐3‐phosphate dehydrogenase decreased sharply.

The present results are discussed in relation to data in the literature on the increased activities of cyclic AMP and cyclic GMP phosphodiesterases in hypothyroidism, observations which integrate the relationships between anabolic and catabolic pathways of carbohydrate and lipid metabolism with alterations in hormone sensitivity in hypothyroidism.
Controlled Proteolysis of Flavocytochrome b2
Authors: POMPON Denis , LEDERER Florence
Published: March 2005

It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33‐36000 and 21000, with some modification of catalytic properties, but without destruction of the oligomeric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 °C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also concluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues.

At 37 °C, chymotrypsin digestion yields a heme‐binding core of molecular weight 15000, larger than the already characterized tryptic heme‐binding core by about 40 residues. Although the latter is known to be very similar to trypsin‐solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent‐solubilized cytochrome b5.
Taipoxin, an Extremely Potent Presynaptic Neurotoxin from the Venom of the Australian Snake Taipan (Oxyuranus s. scutellatus)
Authors: FOHLMAN Jan , EAKER David , KARLSSON Evert , THESLEFF Stephen
Published: March 2005

Taipoxin (taipan toxin), purified from the venom of the Australian taipan (Oxyuranus s. scutellatus) by gel filtration on Sephadex G‐75 followed by column zone electrophoresis, is the most lethal neurotoxin yet isolated from any snake venom. The LD50 is 2 μg/kg in the mouse. The main physiological effect is a gradual reduction to complete stop of evoked and spontaneous release of acetylcholine from motor nerve terminals. Intoxicated animals die of asphyxia caused by neuromuscular blockage of the respiratory muscles.

Taipoxin is a moderately acidic sialo‐glycoprotein (pI 5) with a molecular weight of 45600 as calculated from composition data or 46800 as determined by meniscus depletion sedimentation equilibrium. Taipoxin is a 1:1:1 ternary complex of subunits designated α, β and γ which dissociate completely at low pH and high ionic strength or in 6 M guanidine hydrochloride. The dissociation by guanidine at neutral pH is reversible, while the acid‐induced dissociation is not. The α and β components consist of 120 amino acid residues cross‐linked by seven disulfide bridges, whereas the component has 135 residues and eight disulfides.

The very basic (pI < 10) a component contains 13 residues of arginine and is the only subunit displaying lethal neurotoxicity (mouse LD50∼ 300 μg/kg): The neutral β fraction was separated by ion‐exchange chromatography into two iso‐component, β1 and β2, which differ slightly in amino acid composition. The very acidic γ component contains all of the carbohydrate, which includes 4‐5 residues of sialic aid. The three subunits are homologous in sequence although the γ component is eight residues longer on the N‐terminus and must also contain extra amino acids elsewhere
The Arrangement of 18‐S and 28‐S Ribosomal Ribonucleic Acids within the 40‐S Precursor Molecule of Xenopus laevis
Authors: SCHIBLER Ueli , HAGENBÜCHLE Otto , WYLER Tony , WEBER Rudolf , BOSELEY Paul , TELFORD John , BIRNSTIEL Max L.
Published: March 2005

The arrangement of 18‐S rRNA and 28‐S rRNA within their 40‐S common precursor molecule (pre‐rRNA) of Xenopus laevis was investigated by electron microscopic analysis of secondary structure of nascent pre‐rRNA chains of oocytes, and by 5′‐end analysis of 18‐S rRNA and 28‐S rRNA hybridized to the EcoRI fragment of rDNA cloned as plasmid pCD42.

Secondary structure mapping of phenol‐extracted RNA from nucleolar cores revealed complete pre‐rRNA chains of molecules at various stages of processing and pre‐rRNA molecules apparently lacking one end. In this latter group, which was regarded as representing nascent chains, more than 90% of the molecules had no 28‐S rRNA region. This shows that the 28‐S rRNA sequence is transcribed after the 18‐S rRNA region and hence must be located nearer to the 3′ end of the pre‐rRNA molecule.

For 5′ end‐group determination [3H]uridine‐labelled 18‐S rRNA and 28‐S rRNA were hybridized, as fragments of about 200 nucleotides. to the plasmid pCD42 containing coding sequences for four‐fifths of the 18‐S rRNA sequence, the external transcribed spacer, the non‐transcribed spacer and a tenth of the 28‐S rRNA sequence. The RNA was recovered from the hybrids and analyzed for uridine 3′,5′‐bisphosphate (pUp) after alkaline hydrolysis. The pUp content of the hybridized 18‐S rRNA fragments was 20‐fold‐higher than in those of 28‐S rRNA, thus demonstrating that the 5′ end of the 18‐S rRNA is located next to the external spacer region.

From these results it is concluded that the 18‐S rRNA is located close to the 5′ end of the 40‐S pre‐rRNA molecule.
Organisation of Genes for Ribosomal RNA in Physarum polycephalum
Authors: MOLGAARD Harald V. , MATTHEWS Harry R. , BRADBURY E. Morton
Published: March 2005

Physarum polycephalum nucleolar satellite DNA has been analysed by restriction enzyme digests and hybridisation to ribosomal RNA. The nucleolar DNA is isolated as molecules of molecular weight 39 × 106. The positions of sites of endo R · EcoRl and endo R · HindIII digestion have been determined accurately in the whole molecule. The endo R · EcoRI sites and one endo R ‐ Hind III site are within the 26‐S rRNA complementary sequence and the other endo R · HindIII site is within the 19‐S rRNA complementary sequence. The sites are arranged symmetrically about the centre of the molecule as if it were a palindrome.
6‐Methylsalicylic Acid Synthetase from Penicillium patulum
Authors: DIMROTH Peter , RINGELMANN Erika , LYNEN Feodor
Published: March 2005

The specificity of 6‐methylsalicylic acid synthetase with respect to the priming substrate was studied. If acetyl‐CoA was replaced by propionyl‐CoA 6‐ethylsalicylic acid was synthesized. The rate of this synthesis was about l3% that of 6‐methylsalicylic acid synthesis in presence of acetyl‐CoA.

6‐Methylsalicylic acid synthetase contains an acetyl transferase activity as demonstrated by the transfer of acetyl residues from acetyl‐CoA to pantetheine. This transferase also catalyses propionyl transfer but only with an about 13 times slower rate.

Analogous to fatty acid synthetase, treatment with iodoacetamide converts 6‐methylsalicylic acid synthetase into a malonyl‐CoA decarboxylase. Under certain conditions the iodoacetamide‐treated enzyme could catalyse 6‐methylsalicylic acid formation from malonyl CoA and NADPH without the external addition of acetyl CoA.
Subcellular Distribution and Response to Gastrointestinal Hormones of Adenylate Cyclase in the Rat Pancreas Partial Purification of a Stable Plasma Membrane Preparation
Authors: SVOBODA Michal , ROBBERECHT Patrick , CAMUS Jean , DESCHODT‐LANCKMAN Monique , CHRISTOPHE Jean
Published: March 2005

The subcellular distribution of adenylate cyclase activity in rat pancreatic homogenates was examined after differential centrifugation. Divalent cations exerted significant effects on this distribution. In addition, the ratio of adenylate cyclase activities in the presence of the C‐terminal octapeptide of cholecystokinin‐pancreozymin and secretin was lower in the crude ‘mitochondrial’ fraction than in ‘microsomal’ fractions. This difference was due to the lability of cholecystokinin‐pancreozymin receptors compared to secretin receptors. The Km app of activation was affected more than the V by this lability. Such a degradation of cholecystokinin‐pancreozymin receptors was markedly delayed by isolation and storage in the presence of a phospholipid mixture.

A simple, reasonably rapid (6 h), and easily reproducible method was developed to prepare a stable semi‐purified plasma membrane fraction, characterized by a 10‐fold increase in the specific activity of adenylate cyclase with respect to the whole homogenate. At variance with data obtained on crude subceilular fractions, the V of adenylate cyclase activity observed in this preparation, under maximal concentration of the C‐terminal octapeptide of cholecystokinin‐pancreozymin, was higher than that obtained with secretin or the vasoactive intestinal polypeptide
Kinetic Transients in the Reduction of Aldehydes Catalysed by Liver Alcohol Dehydrogenase
Authors: KVASSMAN Jan , PETTERSSON Gösta
Published: March 2005

The transient‐state kinetics of enzymic reduction of acetaldehyde and benzaldehyde by NADH, cätalyzed by horse liver alcohol dehydrogenase, have been examined under single‐turnover conditions, obtained by carrying out reactions either with limiting amounts of enzyme in the presence of 20 mM pyrazole or with limiting amounts of substrate. Analysis of the variation with substrate, coenzyme, and enzyme concentrations of amplitudes and time constants for the exponential transients observed at 328 nm and 300 nm shows that the kinetics of enzymic aldehyde reduction are qualitatively and quantitatively consistent with the relationships derived in the preceding paper for an ordered ternary‐complex mechanism involving identical and independent catalytic sites. It is concluded that there is no evidence whatsoever for the kinetic significance of a half‐of‐the‐sites reactivity or any other kind of subunit interaction in the liver alcohol dehydrogenase System. The biphasic transients observed at 328 nm for the reduction of aromatic aldehydes such as benzaldehyde are a normal kinetic characteristic of the ordered ternary‐complex mechanism, being attributable to accumulation of the ternary enzyme · NAD · product complex when product dissociation from this complex is slow in comparison to its formation by ternary‐complex interconversion
The Structural Basis for the Heterogeneity of Chick Down Feather Keratin
Authors: WALKER Ian D. , ROGERS George E.
Published: March 2005

The amino acid sequences of prominent tryptic peptides of unfractionated carboxymethylated down feather keratin were determined by the dansyl‐Edman procedure and their arrangement within the intact primary structure deduced by comparison with closely homologous components of emu and gull feather keratin. The presence of multiple amino acid interchanges in different down feather keratin chains was inferred. Most or all keratin chains possessed amino‐terminal and carboxyl‐terminal regions, rich in half cystine, and a large internal region devoid of half‐cystine and rich in hydrophobic amino acids. The size of all down feather keratin chains was conserved and the sequence data were compatible with all chains having the same number of amino acid residues.

The amino acid substitutions reflected the presence in the chick genome of multiple keratin genes differing from one another by one or a few nucleotide changes.
Antibody to Rheumatoid Synovial Collagenase
Authors: WOOLLEY David E. , CROSSLEY Malcolm J. , EVANSON John M.
Published: March 2005

An Immunoglobulin G antibody has been isolated from the sera of sheep immunised with purified synovial collagenase obtained from tissue culture medium of rheumatoid synovium.

In double diffusion gels and on immunoelectrophoresis the antibody gave a single precipitin line when run against crude and pure synovial enzyme preparations. Selective adsorption of collagenase from concentrated svnovial tissue culture medium eliminated the precipitin reponse. No immunoprecipitation was observed with whole serum or synovial tissue homogenates in double diffusion gels.

Incubation of immune immunoglobulin G with synovial collagenase resulted in enzyme inhibition and the formation of immunoprecipitates. Fab′ fragments of the antibody were similarly inhibitory but gave no precipitin response. Non‐specific neutral proteinase activity released bysynovial explants was not inhibited by antibody.

Human collagenases derived from gastric mucosa, cornea, and skin were inhibited by the antibody and produced precipitin lines on immunodiffusion showing a reaction of complete identity with the synovial enzyme. Granulocyte collagenase preparations did not react with the antibody.

The non‐human collagenases derived from rabbit cornea and dog gingiva were inhibited by the antibody but those from guinea pig skin and the bacterium Clostridium histolyticum were not. None of these collagenases gave precipitin responses on immunodiffusion against the antibody.

Synovial enzyme preparations inhibited or denatured by chemical reagents retained their capacity to react with the antibody, whereas inhibition by the serum proteins α2‐macroglobulin or β1‐anticollagenase masked the antigenicity of the enzyme as judged by immunodiffusion experiments. These observations and their relevance to the use of the antibody in elucidating the physiological role of collagenase are discussed.