bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfhydryl Reagents Prevent Dephosphorylation and Proteolysis of Histones in Isolated HeLa Metaphase Chromosomes
Authors: PAULSON James R.
Published: July 2010

It has been shown by D'Anna et al. [Nucleic Acids Res. 5, 3195–3207 (1978)] that histones H1 and H3, which are highly phosphorylated during mitosis in mammalian cells, become rapidly dephosphorylated during conventional metaphase chromosome isolation procedures.

We show here that this dephosphorylation can be completely prevented by including sulfhydryl reagents, such as p‐chloromercuriphenyl sulfonate or 5,5′‐dithiobis(2‐nitrobenzoate), in the chromosome isolation buffers. These reagents also efficiently inhibit the endogenous proteases present in isolated HeLa chromosomes and nuclei.
The Limitations of Chlorotetracycline as a Fluorescent Probe of Divalent Cations Associated with Membranes
Authors: GAINS Nigel
Published: July 2010

The interaction of chlorotetracycline with magnesium chloride, detergents and ATP and ADP has been investigated. At various pH the fluorescence of chlorotetracycline, or the chlorotetracycline complex, is enhanced in the presence of the negatively charged detergent sodium dodecylsulphate, the uncharged detergent Triton X‐100, or the positively charged detergent cetyltritnethylammonittm bromide. Also a ternary fluorescent complex is formed between chlorotetracycline, magnesium and ATP, or ADP. In a biological membrane, it would not seem possible to distinguish between the fluorescence signal arising from chlorotetracycline acting as a probe of divalent cations bound to specific sites on the membrane and that arising from the complex of chlorotetracycline with divalent cations acting as a probe of neutral or negative regions of the membrane.
The Metabolic Fate of Mitochondrial Hydrogen Peroxide
Authors: NOHL Hans , JORDAN Werner
Published: July 2010

Mitochondrial H2O2formation is not in equilibrium with defence mechanisms that counteract an accumulation of H2, in rat‐heart cell.

A model for the accumulation kinetics is proposed which is consistent with the data presented.

Four different pathways of H2O2metabolism are described in rat‐heart mitochondria. The major site for metabolic branching of H2O2Via different routes was found to be the mitchondrial catalase.

Glutathione (GSH) peroxidase accounts for only 15% of intramitochondrial H2O2metabolism, while catalase‐mediated destruction is four times more rapid.

Catalase activity is limited by its structural compartmentation in the matrix, while GSH peroxidase activity was found to be dependent on the availability of free GSH.

Catalase was shown to protect rat‐heart mitochondria from upsetting redox states of GSH and pyridine nucleotides following H2O2, decomposition by GSH peroxidase.

Computer simulations of experimental data Suggest the existence of a third sink for mitochondrial H2O2, possibly due to mitochondrial formation of OH′ radicals; another fraction of the H2O2matrix pool may cross the mitochondrial membrane and accumulate in the cytosol.
Purification and Properties of a DNase Inhibitor from Nicotiana tabacum Cell Cultures
Authors: SZOPA Jan , WAGNER Karl G.
Published: July 2010

Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE‐cellulose and gel filtration resulted in a homogeneous protein (Mr= 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1: 1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor. DNase‐I complex have been calculated. This interaction is strongly temperaturedependent; at 20° C and 26° C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.
Localization of Phosphoproteins and of Protein Kinases in Chromatin from Hepatoma Tissue‐Cultured Cells
Authors: KITZIS Alain , TICHONICKY Lydie , DEFER Nicole , KRUH Jacques
Published: July 2010

An important role in the control of gene expression has been attributed to phosphoproteins present among chromatin, non‐histone proteins. In a previous work we have shown that at least part of these phosphoproteins are associated with nucleosomes. In this work we wanted to establish whether this association occurs with all nucleosomes or with the nucleosomes present in fragments preferentially released by a mild micrococcal nuclease digestion, which originated essentially from active parts of chromatin.

Phosphoproteins were labelled in vivo by incubating hepatoma tissue‐cultured cells with [32P]phosphate and chromatin was submitted to a limited micrococcal nuclease digestion. The released fragments were fractionated by preparative gel electrophoresis. [32P]Phosphoproteins were essentially found in the smallest released fragments; monomers and dimers of nucleosomes. The same result was obtained when the phosphoproteins were labelled in vitro by incubating each fragment obtained by the preparative electrophoresis in the presence of [γ‐32P]ATP. It indicates that part of the protein kinase activity was strongly bound to the particles. The bound phosphoproteins were analysed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Two main polypeptides were characterized: phosphopeptide a, Mr 41000, present in all small fragments; phosphopeptide b, Mr 31000, present in all small fragments, except in the fastest moving nucleosomes.

Phosvitin kinase was found associated with the small released fragments, its specific activity was by far the highest in the fraction which includes the dieters of nucleosomes.

It is concluded that phosphoproteins and protein kinases are associated with the nucleosomes of the active parts of chromatin, which suggests a role of these proteins in the control of gene expression.
The Neutral and Acidic Glyceroglucolipids from Gastric Mucous Barrier of Rat‐Stomach Antrum, Body and Forestomach
Authors: SLOMIANY Bronislaw L. , GALICKI Nina I. , KOJIMA Koichi , SLOMIANY Amalia
Published: July 2010

Distribution of glyceroglucolipids in the mucous barrier of rat‐stomach antrum, body and forestomach was investigated. Surface mucus lining and preformed intracellular mucus were obtained by perfusion in vivo of the ligated stomach compartments with saline and 2 M NaCl. Lipids were extracted from dialyzed and lyophilized samples, and separated on DEAE‐Sephadex into neutral and acidic fractions. The glyceroglucolipids contained in each fraction were quantified and then separated into individual components by thin‐layer chromatography. Significant differences in the concentration of glyceroglucolipids in the separated areas of the stomach were found. The content of glyceroglucolipids in the antral portion of the stomach was five‐times greater than that of the forestomach. The level of sulfated glyceroglucolipids was eight‐times greater in the antrum as compared to the body and four‐times greater as compared to the forestomach. One sulfated glyceroglucolipid was present in antral perfusates, whereas the body and forestomach contained two additional acidic glyceroglucolipids. Based on data of partial acid and alkaline methanolyses, oxidation with periodate and chromium trioxide, and methylation of the native and desulfated compound, the structure of antral glyceroglucolipid is proposed to be: SO3H(→6)Glc(α1→6)Glc(α1→6)Glc(α1→6)Glc(α1→3)‐1(3)‐O‐alkyl‐2‐O‐acylglycerol. The neutral glyceroglucolipid fractions from each area of the stomach were similar in composition and only variations in their concentration were observed.

The data obtained suggest that the sulfated glyceroglucolipids are involved in the protection of the gastric mucosa of the antrum, since their concentration is greatest in this most acidic portion of the stomach which is also most often exposed to duodenal reflux.
The Primary Structure of the Constant Part of μ‐Chain‐Disease Protein BOT
Authors: MIHAESCO Edith , BARNIKOL‐WATANABE Shitsu , BARNIKOL Heinz Ulrich , MIHAESCO Constantin , HILSCHMANN Norbert
Published: July 2010

The complete primary structure of the constant part of the u‐chain‐disease protein, BOT, was established. It includes the whole CH2, CH3 and CH4 domains. Two amino acid changes were found, at positions 309 (Ser→Gly) and 333 (Val→ Gly) (GAL numbering). In two additional monoclonal μ chains (SCO and CO), the same positions showed an amino acid variability. From these data it may be concluded that four types of μ chains exist in the human: (1) GAL type with Ser‐309 and Val‐333; (2) OU type with Gly‐309 and Val‐333; (3) SCO type with Ser‐309 and Gly‐333; (4) BOT/CO type with Gly‐309 and Gly‐333. The meaning of this molecular polymorphism is discussed.
Globin‐Chain Affinity Chromatography on Sepharose‐Haptoglobin a New Method of Study of Hemoglobin Synthesis in Reticulocytes, in Bone Marrow and in Colonies of Erythroid Precursors
Authors: TSAPIS Andréas , HINARD Nicole , TESTA Ugo , DUBART Anne , VAINCHENKER William , ROUYER‐FESSARD Philippe , BEUZARD Yves , ROSA Jean
Published: July 2010

In the present work we have developed an affinity chromatography system, using haptoglobin bound covalently to Sepharose 4B, to purify hemoglobin from soluble non‐heme proteins. Sepharosehaptoglobin specifically binds hemoglobin. It exhibits the same characteristics in its interactions with hemoglobin and α or β hemoglobin chains as does haptoglobin in solution. Globin chains can be eluted from the Sepharose‐haptoglobin after removal of the heme. This method has allowed accurate measurements of globin‐chain synthesis in blood and bone marrow samples and in culture of early erythroid precursors.
The Binding of Oxidised Coenzyme to Bovine‐Liver Glutamate Dehydrogenase Studied by Circular‐Difference Spectroscopy
Authors: BAYLEY Peter M. , O'NEILL Kevin T. J.
Published: July 2010

The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high‐sensitivity circular dichroism (CD) difference spectroscopy.

The CD of the bound coenzyme in the binary complex E · NAD closely resembles that of bound ADP, although the affinity is much lower, being 350‐fold less for NAD+ at 20°C in 0.1 M phosphate, pH 7.

A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E · NAD.

Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1:1 complex E · NAD with Kd= 0.72 mM in a simple binding process without positive or negative cooperativity.

NADP+ binding is more than 10‐fold weaker than NAD+ binding.

From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II).

Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E · NAD‐glatarate. This process occurs with low affinity and is probably also located at the adenine regulatory site.

This second‐site binding of NAD+ may contribute to the phenomena of non‐Michaelis‐Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit‐subunit cooperative interactions.
Non‐specific Biosynthesis of Hopane Triterpenes by a Cell‐Free System from Acetobacter pasteurianum
Authors: ROHMER Michel , ANDING Claude , OURISSON Guy
Published: July 2010

A cell‐free syqtem from the bacterium Acetobacter pasteurianum was incubated with [12‐3H]‐squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin‐layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum.

The same cell‐free system transformed (RS)‐2,3‐epoxy‐2,3‐dihydro‐[12,13‐3H]squalene into labelled 3α‐hydroxyhop‐22(29)‐ene, 3β‐hydroxyhop‐22(29)‐ene, hopane‐3α,22‐diol and hopane3β,22‐diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3‐epoxy‐squalene, into 3‐hydroxyhopanoids normally absent in the bacterium.

The 3α‐hydroxy and 3β‐hydroxyhopanoids could have been enzymatically interconverted via the 3‐oxo compound. Synthetic racemic (RS)‐2,3‐epoxy‐2,3‐dihydro‐[3‐3H]squalene was incubated and gave rise to 3‐3H‐labelled 3α and 3β‐hydroxyhopanoids. This excludes an isomerization via a 3‐oxo compound which would give unlabelled 3‐hydroxyhoparoids.

In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, saualene, by the corresponding cpoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.
Metabolism of NAD and N1‐Methylnicotinamide in Growing and Growth‐Arrested Cells
Authors: JOHNSON George S.
Published: July 2010

Nicotinamide is metabolized primarily into NAD and N1‐methylnicotinamide in cultured cells of normal rat kidney. Tne metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1‐Methylnicotinamide levels are 1.5‐2‐fold elevated to cells growth‐arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than a decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serumstarved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20‐fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds.

In medium containing normal amounts of nicotinamide, the apparent first‐order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h‐1. Labeled N1‐methylnicotinamide is not metabolized, but rather is excreted into the medium with a first‐order rate constant of 3.9 h−1. The rate of loss of label from NAD, but not from N1‐methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1‐methylnicotinamide.

The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADPribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1‐methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1‐methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.
Benzil, a Potent Activator of Microsomal Epoxide Hydrolasein vitro
Authors: SEIDEGÅRD Janeric , DEPIERRE Joseph W.
Published: July 2010

Benzil was found to be a very potent activator of microsomal epoxide hydrolase activity (measured with styrene oxide as substrate)in vitro. The activating effect was uncompetitive and benzil causes approximately ninefold increases in both the apparentVand the apparentKmof the enzyme(s). The half‐maximal effect on activity was obtained as a 0.3 mM concentration of benzil.

The activating effect obtained with benzil was found to be very specific, since a variety of structurally related compounds had little or no effect on microsomal epoxide hydrolase activity. In order to obtain indications for the existence of more than one microsomal epoxide hydrolase the effect of benzil on this activity from rats induced with phenobarbital, 3‐methylcholanthrene, 2‐acetylaminofluorene,trans‐stilbene oxide, and benzil was tested. The differences observed were minor.
Anti‐mitogenic Function of Interferon‐Induced (2′‐5′)Oligo(adenylate) and Growth‐Related Variations in Enzymes that Synthesize and Degrade This Oligonucleotide
Authors: KIMCHI Adi , SHURE Helen , REVEL Michel
Published: July 2010

Addition of (2′‐5′)ApApA to concanavalin‐A‐stimulated mouse spleen lymphocytes strongly inhibits the large increase in RNA and protein synthesis which takes place 24–48 h after stimulation. The inhibitory effect on protein synthesis precedes the effect on RNA synthesis and takes at least 6 h to be detected. Histone synthesis is preferentially inhibited at 48 h. No effect on protein synthesis was detected in unstimulated resting lymphocytes, or in stimulated lymphocytes during the first 24 h after concanavalin A treatment. The anti‐mitogenic effect of the (2′‐5′)oligo(adenylate) seems to result, therefore, from inhibition of protein synthesis taking place before initiation of DNA replication. The mitogenic stimulus produced by the lectin enhances, in lymphocytes, the level of the 2′‐phosphodiesterase which degrades (2′‐5′)oligo(adenylate). Enhancement of the 2′‐phosphodiesterase was also observed after serum stimulation of confluent monkey kidney cells. Furthermore, the ratio of (2′‐5′)oligo(adenylate) synthetase to 2′‐phosphodiesterase is ten‐times lower in fast‐growing kidney cells than in quiescent serum‐starved cells. A model for the role of (2′‐5′)oligo(adenylate) synthesis and degradation in the regulation of cell proliferation by interferon and by mitogens is presented.
Specificities of Limulin and Wheat‐Germ Agglutinin Towards Some Derivatives of GM3 Gangliosides
Authors: MAGET‐DANA Régine , VEH Rüdiger W. , SANDER Michael , ROCHE Annie‐Claude , SCHAUER Roland , MONSIGNY Michel
Published: July 2010

Lipid vesicles containing derivatives of GM3 ganglioside (II3‐N‐acetylneuraminosyllactosyl ceramide) were used to study the specificities of two lectins (limulin and wheat germ agglutinin) towards N‐acetyl neuraminic acid and N‐glycoloylneuraminic acid and some of their natural and chemically modified derivatives.

The extent of the lectin binding to the gangliosides was related to the aggregation process of the lipid vesicles which was monitored as an absorbance increase.

Limulin binds specifically to lipid vesicles containing N‐glycoloyl derivatives of GM3. The hydroxyl group at C‐4 and the carboxyl group of neuraminic acid have to be free for the binding to limulin. The side chain of neuraminic acid is not involved in the binding site of limulin.

Wheat germ agglutinin binds to GM3 ganglioside only when the hydrophilic tail of the neuraminic group is cut off (C7 analogues). The acetamido group but not the carboxyl group is involved in the binding to wheat germ agglutinin. The wheat‐germ‐agglutinin‐induced aggregation of vesicles containing derivatives of GM3‐ganglioside is dependent on the pH, on the ionic strength and on the presence of Ca2+ ions. The dependence on ionic strength and Ca2+ is a consequence of the electrostatic repulsion of the vesicles.

The wheat‐germ‐agglutinin‐induced aggregation process of vesicles containing any suitable GM3‐ganglioside derivative was reversed by the addition of N‐acetylglucosamine showing that the N‐acetylneuraminic acid derivatives bind to the N‐acetylglucosamine binding site.
A Cysteine‐Rich Collagenous Protein from Bovine Placenta
Authors: JANDER Renate , RAUTERBERG Jürgen , VOSS Bruno , von BASSEWITZ Dirk B.
Published: July 2010

A high‐molecular‐weight collagenous protein was isolated, after limited pepsin digestion, from the endometrial villi of bovine placenta; it resembled the protein isolated from human aortas by Chung et al. [Chung, E., Rhodes, R. K. and Miller, E. J. (1976) Biochem. Biophys. Res. Commun. 71, 1167–1174] and from human placenta by Furuto and Miller [Furuto, D. K. and Miller, E. J. (1980) J. Biol. Chem. 255, 290–295]. After denaturation and reductive cleavage of disulfide bridges three chains with Mr of 40000–55000 were liberated. Two of the chains could be separated by ion‐exchange chromatography; the third chain aggregated under non‐denaturing conditions and could be isolated by sequential molecular‐sieve chromatography in denaturing and non‐denaturing buffers. Their amino acid composition resembled that of basement membrane collagens with respect to the high content of completely glycosylated hydroxylysine but differed with respect to their relatively low hydroxyproline content and high values of cysteine, tyrosine and glucosamine. A relatively low glycine content indicated regions of non‐collagenous structure. Reduction under non‐denaturing conditions and another pepsin treatment removed non‐collagenous regions containing mainly hydrophobic amino acid residues. The denaturation temperature of the component was considerably reduced by this treatment, indicating stabilization of triple‐helical structures by disulfide bridges in the native molecule.

Fibrillar aggregates were precipitated with ATP which showed in the electron microscope a typical crossstriation pattern different to those of segment‐long‐spacing crystallites obtained from other collagen types. Immunofluorescence tests demonstrated the protein in blood vessels and interstitial areas of the renal medulla but not in basement membranes of glomeruli and tubuli.
Studies on the Posttranscriptional Site of cAMP Action in the Regulation of the Synthesis of Tyrosine Aminotransferase
Authors: SNOEK Gerry T. , VAN DE POLL Klaas W. , VOORMA Harry O. , VAN WIJK Roel
Published: July 2010

Synthesis of l‐tyrosine: 2‐oxoglutarate aminotransferase (EC 2.6.1.5) can be induced by N6,O2′‐dibutyryladenosine 3′,5‐monophosphate (Bt2cAMP) in Reuber H35 cell cultures. Actinomycin D fails to block this induction which indicates a target for Bt2cAMP at a posttranscriptional level. We have determined the influence of Bt2cAMP on several translational events during the tyrosine aminotransferase synthesis with the following results. (1) The number of nascent tyrosine aminotransferase chains increased, whereas no effect was measured on the number of nascent total protein chains. (2) The rate of elongation along the tyrosine aminotransferase mRNA and total mRNA is not enhanced by Bt2cAMP. (3) The induced synthesis of tyrosine aminotransferase is more sensitive to the inhibition of elongation. We conclude from our results that Bt2cAMP induces the synthesis of tyrosine aminotransferase by an increase in the rate of initiation on the tyrosine aminotransferase mRNA.
Competition between Two Pathways for Sugar Uptake by the Phosphoenolpyruvate‐Dependent Sugar Phosphotransferase System in Salmonella typhimurium
Authors: SCHOLTE Bob J. , POSTMA Pieter W.
Published: July 2010

The interaction between the two pathways for glucose entry via the phosphoenolpyruvate: sugar phosphotransferase system, i.e. via enzyme II‐A/II‐B and enzymes II‐BGlc/IIIGlc, was studied in Salmonella typhimurium. Thio‐β‐d‐glucoside and 5‐thio‐d‐glucose were shown to be substrates of P‐pyruvate: sugar phosphotransferase specific for enzyme II‐BGlc both in intact cells and in toluene‐treated cells of S. typhimurium. The activity of the II‐A/II‐B pathway was strongly inhibited by the presence of II‐BGlc substrates. It is concluded that the two pathways compete for phosphoryl groups provided by P‐pyruvate, and that under the conditions tested the flow of phosphoryl groups through enzyme I/HPr is the rate‐limiting step in vivo of activity of the pathways studied. The results corroborate the proposed mechanism of the regulatory function of the P‐pyruvate: sugar phosphotransferase system which predicts a net dephosphorylation of components of the P‐pyruvate: sugar phosphotransferase in the presence of a substrate of P‐pyruvate : sugar phosphotransferase.
Cathepsin D: Specificity of Peptide‐Bond Cleavage in Type‐I Collagen and Effects on Type‐III Collagen and Procollagen
Authors: SCOTT Paul G. , PEARSON Harold
Published: July 2010

Cathepsin D, purified from bovine thymus, has a limited proteolytic effect on types I and III bovine collagens. The α1 (I) chain was cleaved in native or denatured collagen only within the carboxy‐terminal extrahelical sequence, the major site being between residues C6 (Leu) and C7 (Ser). The α2 chain was unaffected in native collagen but was slowly cleaved between residues 782 (Phe) and 783 (Leu) in the denatured form. Cleavages, at 45° C, in type III collagen occur within the extra‐helical amino‐terminal sequence, on the carboxyterminal side of the lysine residue involved in intermolecular cross‐linking. All three sites of action are within sequences of general hydrophobic character.

The very restricted cleavage of peptide bonds in denatured collagens can be ascribed to the infrequent occurrence of groupings of more than two hydrophobic residues and to the high content of the conformation limiting residues proline and hydroxyproline.

The previously demonstrated failure of cathepsin D to solubilize a representative proportion of type III collagen from the fibres of bovine skin collagen (P. G. Scott and C. H. Pearson (1978) Biochem. Soc. Trans. 6, 1197–1199] may be explained by lack of ability of the enzyme to act on this collagen at 25° C, in such a manner as to separate molecules joined by intermolecular cross‐links involving the amino‐terminal extrahelical region of the molecule.
Decrease in the S1 Protein of 30‐S Ribosomal Subunits in Polyamine‐Requiring Mutants of Escherichia coli Grown in the Absence of Polyamines
Authors: IGARASHI Kazuei , KASHIWAGI Keiko , KISHIDA Kazuo , KAKEGAWA Tomohito , HIROSE Seiyu
Published: July 2010

The reason for the decrease of polypeptide‐synthetic activity of 30‐S ribosomal subunits obtained from two polyamine‐requiring mutants of Escherichia coli, grown in the absence of polyamines, has been studied by analyzing the total and split proteins of 30‐S subunits by disc gel and slab get electrophoreses. It was concluded that the decrease of S1 protein in 30‐S subunits was responsible for the decrease of polypeptide synthesis in polyamine‐requiring mutants of E. coli grown in the absence of polyamines.
A Catalytic Mechanism for Benzylamine Oxidase from Pig Plasma
Authors: YADAV Kapil D. S. , KNOWLES Peter F.
Published: July 2010

Ammonium ion is shown to decrease the rate constants for Schiff's base formation and formation of a reduced intermediate during the catalytic cycle of benzylamine oxidase from pig plasma. The rate constant for reoxidation of the reduced intermediate is also inhibited whilst the rate constant for conversion of the oxidised enzyme form back to native enzyme is stimulated by ammonium ion.

Ammonium ion changes the electron paramagnetic resonance spectrum of the cupric centres in the enzyme, indicating that ammonia binds to the copper.

A catalytic mechanism for benzylamine oxidase is proposed on the basis of these and other results. This mechanism includes a novel step in which a hydroxyl coordinated to copper acts as a nucleophyle to facilitate hydride ion transfer to oxygen during the reoxidation process.
Isolation and Characterization of Binder IIIA, a New Protein Which Binds Corticosteroid Anions
Authors: STEEGER Joseph R. , LITWACK Gerald
Published: July 2010

A new protein binding corticosteroid metabolites has been purified over 300‐fold from liver cytosols of adrenalectomized rats, treated for 45 min in vivo with [1,2‐3H]cortisol. Purification was accomplished by column chromatography on Sephadex G‐25, DEAE‐Sephadex A‐50, Sephadex G‐75, and hydroxylapatite. The protein has a Stokes radius of 2.27 nm by gel filtration and an apparent sedimentation coefficient of 3.0 S by sucrose gradient centrifugation. The calculated molecular weight is 30700. The bound steroid was extracted and has been shown by Sephadex LH‐20 chromatography to be a monosulfate derivative of cortisol.

Using liver cytosol from adrenalectomized rats pretreated in vivo for 45 min with [1,2‐3H]cortisol plus 1000‐fold excess competing steroid, cortisol derivatives and progesterone were shown to be the most active competitors. Testosterone and 17β‐estradiol were least active as competitors. The synthetic steroids, dexamethasone and triamcinolone, produced little or no competition. The protein has been named corticosteroid‐anion binder IIIA in keeping with its elution position from a DEAE‐Sephadex A‐50 column, compared to other binding proteins.

Binder IIIA has been separated chromatographically from the glutathione S‐transferases (including ligandin) and protein z described by Arias [Levi, A. J., Gatmaitan, Z. and Arias, I. M. (1969) J. Clin. Invest. 48, 21856–21866], both of which have been shown to bind anionic metabolites. It has been resolved from the activities of transcortin, cortisone 5β‐reductase, and 3α‐hydroxysteroid dehydrogenase.
Molecular Properties of Two Mutant Species of the Elongation Factor Tu
Authors: VAN DER MEIDE Peter H. , Jan DUISTERWINKEL Feitse , DE GRAAF J. Martien , KRAAL Barend , BOSCH Leendert , DOUGLASS James , BLUMENTHAL Thomas
Published: July 2010

The molecular properties of two mutant species of the elongation factor Tu (EF‐Tu), derived from either tuf A or tufB, have been studied. One, designated EF‐TuAR, is the product of a kirromycin‐resistant tufA gene. The other designated EF‐TuBo is a tufB product and is present in a kirromycin‐resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF‐TuAR species. EF‐TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tufB gene has been inactivated by an insertion of the bacteriophage Mu. EF‐TuBo has been isolated from LBE 2012 together with EF‐TuAR in a 1:1 mixture. Fractionation of this mixture on DEAF‐Sephadex A‐50 resulted in an enrichment of EF‐TuBo of about 80%. The properties of EF‐TuAR and EF‐TuBo have been compared to those of a kirromycin‐sensitive species designated EF‐TuAS, which was isolated from LBE2045 by transduction of wild‐type tufA.

We show here that all three EF‐Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF‐Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF‐TuAS (wild‐type tufA gene product) to be retained on, and thus block, the ribosome. (b) EF‐TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EFTuBo fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF‐TuAR (LBE2012) is kirromycin resistant.
A Mutant Elongation Factor Tu which Does Not Immobilize the Ribosome upon Binding of Kirromycin
Authors: DUISTERWINKEL Feitse Jan , DE GRAAF J. Martiea , SCHRETLEN Peter J. M. , KRAAL Barend , BOSCH Leendert
Published: July 2010

In the accompanying paper we have shown that polypeptide synthesis sustained by the mutant elongation factor EF‐TuBo is inhibited by kirromycin. Here we have searched for the primary site of inhibition in the elongation cycle. It is demonstrated that in the presence of the antibiotic EF‐TuBo can form a complex with aminoacyl‐tRNA and GTP and that the complex is able to bind to ribosomes programmed with poly(U). Like its wild‐type counterpart, EFTuBo· GDP can form a quaternary complex with aminoacyl‐tRNA and kirromycin but, unlike the wild‐type quaternary complex, the mutant complex fails to associate with the ribosome. This explains the recessive nature of the tufB mutation in cells producing kirromycin‐resistant EF‐TuA and EF‐TuBo. It also suggests a mechanism for the inhibition by kirromycin of EF‐TuBo‐dependent polypeptide synthesis.
Kinetic Comparisons Between Soluble and Membrane‐Bound Glutaminase Preparations from pig Brain
Authors: NIMMO Gillian A. , TIPTON Keith F.
Published: July 2010

The kinetic properties of membrane‐bound glutaminase from pig brain have been compared with those of the soluble enzyme that had been preincubated with either Tris/HCl buffer or Tris/phosphate/borate buffer. The two preparations of the soluble enzyme were similar in the dependence of their activities on pH and in their inhibition by borate and some glutamine analogues. They differed markedly in their responses to phosphate and their inhibition by glutamate. The membrane‐bound enzyme differed from the other preparations in showing a sigmoid dependence of initial velocity on glutamine concentration at higher pH values and being activated by borate ions. The apparently cooperative behaviour that has been previously reported for soluble preparations of glutaminase may have resulted from failure to measure the true initial rate of the reaction. The Km, values for glutamine and the inhibitor and activator constants for glutamate and phosphate respectively were such as to indicate that changes in the concentrations of these compounds might affect the glutaminuse activity in vivo. None of the enzyme preparations was inhibited significantly by ammonium ions at concentrations up to 50 mM. Considerable differences were found between the three preparations in their activation by compounHs containing sulphate or sulphonate groups.
Protein‐Bound Polymeric and Monomeric ADP‐Ribose Residues in Hepatic Tissues
Authors: WIELCKENS Klaus , BREDEHORST Reinhard , ADAMIETZ Peter , HILZ Helmuth
Published: July 2010

Determination of poly(ADP‐ribose) levels was performed by a new procedure involving covalent chromatography of released polymer, degradation to phosphoribosyl AMP and quantification of this specific derivative by a radioimmunoassay. In adult rat liver, about 85 pmol polymeric ADP‐ribose residues/g tissue was found. Similar values were obtained when the determination was carried out by an independent procedure not involving boronate chromatography or sedimentation of DNA.

In adult rat liver, polymeric ADP‐ribose residues amounted to about 1/200 of total monomeric ADP‐ribose residues. Most of the polymeric ADP‐ribose residues were linked to proteins by NH2OH‐sensitive bonds, while mono(ADP‐ribose)‐protein conjugates consisted of about equal amounts of NH2OH‐sensitive and NH2OH‐resistant subfractions.

Poly(ADP‐ribose) levels immediately after birth were similar to the adult status. They decreased, however, by a factor of three at the time of most rapid post‐natal liver growth (day 17). A comparison with the protein‐bound monomeric ADP‐ribose residues indicated independent changes, and therefore presumably independent functions of these monomeric ADP‐ribose residues.
Kinetics of Thyroglobulin Iodination and Thyroid Hormone Synthesis Catalyzed by Peroxidases: The Role of H2O2
Authors: VIRION Alain , POMMIER Jacques , DEME Danièle , NUNEZ Jacques
Published: July 2010

When thyroglobulin iodination by thyroid peroxidase or lactoperoxidase is studied with optimal concentrations of H2O2‐generating system, thyroid hormone synthesis (coupling reaction) begins after a constant lag period which is independent of the concentration of iodide, thyroglobulin or enzyme. Increasing the concentration of the H2O2‐generating system inhibited the iodination reaction whereas the length of the lag period was decreased. When thyroglobulin containing iodinated tyrosine residues but no hormone was incubated with increasing concentrations of iodide and limiting amounts of the H2O2‐generating system the iodination reaction was stimulated and the coupling reaction was inhibited.

To analyze whether the iodination and coupling reactions require different enzyme‐H2O2 species the iodination and coupling reactions and the absorbance at 430 nm (compound II) were measured simultaneously with lactoperoxidase. Under these conditions: (a) the coupling reaction occurred only in the presence of sufficient amounts of H2O2 whereas the level of iodination was the same; (b) an increase in absorbance at 430 nm was observed only when the iodination reaction leveled off, i.e. just before the beginning of the coupling reaction; (c) during the lag period, i.e. the iodination step, the only enzyme species seen in the Soret region was the native enzyme; and (d) adding iodinated thyroglobulin containing no hormone to preformed compound II or III resulted in a prompt coupling reaction. These data suggest that the iodination and coupling reactions are catalyzed by different enzyme‐H2O2 species.

When thyroid peroxidase or lactoperoxidase were preincubated for varying periods of time in the presence of the H2O2‐generating system their activity towards guaiacol decreased progressively: with thyroid peroxidase the Km for guaiacol was increased 10‐fold after 5 min of preincubation and 40‐fold with lactoperoxidase. The activity of the preincubated enzymes was restored by iodide. Preincubated lactoperoxidase had the Soret spectrum of compound III. Similarly, the rate of iodination of thyroglobulin was inhibited with the preincubated enzyme, but increasing the iodide concentration restored the iodinating activity.

The proposal is made that different enzyme‐H2O2 species are the active agents in the catalysis of the iodination and coupling reaction respectively.
Non‐polysomal Poly(A)‐Containing Messenger Ribonucleoproteins of Cryptobiotic Gastrulae of Artemia salina
Authors: SLEGERS Herman , DE HERDT Erik , KONDO Masatoshi
Published: July 2010

Non‐polysomal poly(A)‐containing messenger ribonucleoprotein (mRNP) of Artemia salina has been isolated by thermal chromatography on oligo(dT)‐cellulose in moderate (250 mM) and low (50 mM NaCl and 5 mM MgCl2) ionic strength. The purified particles sedimented between 5 S and 30 S and banded at a density of 1.38–1.40 g/cm3 and 1.26–1.27 g/cm3 in CsCl and sucrose isopycnic centrifugation, respectively. The translatability of the mRNP in a cell‐free system depended on the conditions of isolation. The protein composition of the free mRNP is independent of the conditions used in oligo(dT)‐cellulose chromatography. The proteins have Mr of 87000, 76000, 65000, 50000, 45000, 38000 and 23500. A specific set of proteins is associated with different ribonucleoproteins, although some proteins are present on multiple particles. The main 17 ± 2‐S particle is composed of proteins with Mr of 87000, 76000, 45000 and 38000. Approximately the same proteins were present on free mRNP and mRNP isolated from non‐polysomal mRNP‐ribosome complexes. Poly(A)‐binding proteins have Mr of 38000 and 23500. The 38000‐Mr protein comprised at least 60% of the total mRNP protein. Poly(A)‐binding proteins with Mr of 38000 and 76000 are also present in a free state in the cytoplasm. A relation between the main poly(A)‐binding mRNP protein and the helix‐destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466–6472] is discussed.
Characterization of the α‐Bungarotoxin Receptor in Chick‐Embryo Retina
Authors: BETZ Heinrich
Published: July 2010

Primary cultures and membranes fractions from chick embryo retina bind iodinated α‐bungarotoxin, a highly selective ligand for nicotinic acetylcholine receptors from skeletal muscle and fish electric organ. The binding is saturable with an equilibrium dissociation constant (Kd) of 0.75 ± 0.09 nM. The pseudo‐first‐order rate constant (k+1) for binding at 37°C is 1.76 × 105 M−1s−1, the dissociation rate constant (k−1) at 37°C is 1.15 × 10−1s−1. Nicotinic cholinergic ligands and local anaesthetics inhibit α‐bungarotoxin binding. In the case of carbamoylcholine, the inhibition of binding depends on the time of exposure to this cholinergic agonist. α‐Bungarotoxin has no effect on the carbamoylcholine‐induced stimulation in sodium permeability of cultured retinal neurons. On sucrose density gradients containing Triton X‐100. the toxin binding site sediments with an apparent sedimentation coefficient of 10.5–11 S. Detergent‐solubilized α‐bungarotoxin receptor crossreacts with antisera raised against nicotinic acetylcholine receptors fromTorpedo marmorata. These results are interpreted as indicating that α‐bungarotoxin binds to retinal nicotinic acetylcholine receptors without affecting cholinergic receptor function.
Isolation of Component C4 of Human Complement and Its Polypeptide Chains
Authors: LUNDWALL Åke , MALMHEDEN Ingrid , SJÖQUIST John , STÅLENHEIM Gunnemar
Published: July 2010

Component C4 of human complement was purified from fresh frozen plasma with a yield of 25%, using an initial batch separation with quaternary diethyl‐(2‐hydroxypropyl)aminoethyl—Sephadex followed by column chromatography on DEAE‐cellulose and gel filtration in Sephadex G‐200.

The final product was homogenous according to polyacrylamide gel electrophoresis and immunochemical methods. Low‐speed sedimentation‐equilibrium analyses revealed a molecular weight of 189000, using a value of 0.736 ml/g for the partial specific volume.

The polypeptide chains of reduced and alkylated C4 were separated on DEAE‐Sepharose in the presence of 8 M urea. Gel filtration in Sepharose 4B in 6 M guanidine hydrochloride revealed molecular weights of 88000, 72000 and 32000 for the α, β and γ chain respectively.

The amino acid compositions of component C4 and its constitutive chains were also determined.
A Small‐Angle X‐Ray Scattering Study of the Complex Formation between Elongation Factor. Tu · GTP and Valyl‐tRNAVal1 from Escherichia coli
Authors: ÖSTERBERG Ragnar , SJÖBERG Bo , LIGAARDEN Roar , ELIAS Per
Published: July 2010

The complex formation between elongation factor Tu (EF‐Tu) · GTP and valyl‐tRNAVal1 has bin investigated using the small‐angle X‐ray scattering titration technique. The main species observed is a 1:1 complex with a stability constant log K ≥ 6. The corresponding interaction between EF‐Tu · GTP and non‐aminoacylated tRNA appears to be much weaker with an estimated log K∼4.

The radius of gyration determined for the EF‐Tu · GTP—valyl‐tRNAVal1 complex is larger (R = 3.6 nm) than that of EF‐Tu · GTP (R = 2.5 nm). Likewise, the maximum distance within this complex is larger (Dmax= 12.5 nm) than the one within EF‐Tu · GTP (Dmax= 8.5 nm). These data as well as the p(r) curve are consistent with a multiellipsoid model for the complex. From this model it is indicated that the acceptor stem of tRNA is attached to EF‐Tu and that the anticodon stem and loop protrude into the solution.
Studies on the Actomyosin ATPase and the Role of the Alkali Light Chains
Authors: POPE Brian , WEEDS Alan G. , WAGNER Paul D.
Published: July 2010

Myosin isoenzymes, highly enriched in either alkali 1 or alkali 2 light chains have been prepared by light chain exchange in 4. 7 M ammonium chloride, under conditions where there is minimal loss of ATPase activity. While the actin‐activated ATPase measurements were complicated by a biphasic dependence on actin concentration, the two myosin isoenzymes behaved in a similar manner; at a variety of ionic strength conditions their maximum rates of ATP hydrolysis were nearly identical. Furthermore, under conditions where their Km values could be reliably determined, their apparent affinities for actin in the presence of ATP did not differ greatly. These results suggest that the presence of a particular alkali light chain does not influence the maximum rate of ATP turnover by actomyosin under ionic strength conditions approximating physiological.
Plasma Lipoproteins of Leukemic Guinea Pigs (L2C) Can Regulate Cholesterol Biosynthesis by Lymphocytes of Normal Guinea Pigs
Authors: SAINTE‐MARIE Josette CHUILLON , AUTHIER Marie H. , CAYZAC Monique , PHILIPPOT Jean R.
Published: July 2010

The defect of regulation of cholesterol biosynthesis by leukemic (L2C) guinea pig lymphocytes is not a consequence of serum lipoprotein modifications which would make them unable to participate in the regulatory process. Low density lipoprotein of leukemic animals, in parallel to normal low density lipoprotein, can inhibit the cholesterol biosynthesis by normal cells. Surprisingly, very low density lipoprotein of leukemic animals have the same inhibitory property. Analyses of serum of leukemic animals showed a larger amount of the different lipoprotein fractions (+323% very low density, +27 % low density lipoproteins, the high density lipoprotein staying undetectable in control and leukemic sera) than in normal serum. L2C leukemia produces low density lipoprotein slightly richer in unesterified cholesterol and very low density lipoprotein markedly modified by an increased proportion of unesterified cholesterol, phospholipids and apoprotein B. The inhibitory power of leukemic very low density lipoprotein is discussed by analogy with corresponding power of normal low density lipoprotein which can operate either by the way of binding to the low density lipoprotein receptor or by exchange of unesterified cholesterol between the lipoprotein and the cell.
Sidedness of e− Donation and Stoichiometry of H+ Pumps at Sites II + III in Mitochondria from Rat Liver
Authors: DI VIRGILIO Francesco , POZZAN Marina , AZZONE Giovanni Felice
Published: July 2010

The stoichiometry of the redox H+ pumps at site II and site II+III of the respiratory chain of rat‐liver mitochondria has been determined with three different e− donors: succinate, duroquinol and NADH with duroquinone by‐pass of rotenone inhibition. Succinate provides reducing equivalents to the cytochrome b‐c1 complex from the matrix side. Duroquinol, either added from the outside or obtained from the reduction of exogenous duroquinone by the NADH dehydrogenase, presumably is an intramembrane e− donor to cytochrome b or to the endogenous ubiquinone.

The stoichiometries were determined at steady‐state rates of ion translocation and e− transfer. The ion translocation was followed on the basis of the rates of H+ extrusion and of K+ and Ca2+ uptake. The e− transfer was followed either with the polarographic or with the spectrophotometric technique, i. e. absorbance changes during oxidation of duroquinol to duroquinone. In this latter case N‐ethylmaleimide was used to inhibit the NADH dehydrogenase activity responsible for the re‐reduction of the duroquinone formed during duroquinol oxidation. During operation of sites II+III, with oxygen as e− acceptor, all three substrates tested gave H+/2e− and charge/2e− ratios close to 8. 0. During operation of site II, with ferricyanide as e− acceptor, the H+/2e− ratios were about 4. 0 and the charge/2e− ratios close to 2. 0. The present results support previous reports from our laboratory on the stoichiometries of the redox H+ pumps at sites II and III. Furthermore the equality of the H+ and charge stoichiometry, with substrates having different sides of interaction with complex III suggests that e− donors located on the matrix side or intramembrane (and possibly on the cytoplasmic side) share the same pathway in order to feed electrons into the H+ pump at site II.
Binding of Spermine to tRNATyr Stabilizes the Conformation of the Anticodon Loop and Creates Strong Binding Sites for Divalent Cations
Authors: NÖTHIG‐LASLO Vesna , ŽIVKOVIĆ Tomislav , KUĆAN Željko , WEYGAND‐DURAŠEVIĆ Ivana
Published: July 2010

Electron spin resonance (ESR) spectra of yeast tRNATyr, spin‐labelled in the isopentenyladenosine residue adjacent to the anticodon, were measured as a function of temperature at various spermine/tRNA ratios. The critical temperature, at which a change in the activation energy for spin‐label motion takes place, changes abruptly by almost 10°C upon the addition of the fifth spermine molecule/tRNATyr molecule, indicating a marked stabilization of the anticodon region.

Scatchard plots for Mn2+ binding to tRNATyr in the presence of spermine do not follow theoretically predicted curves for electrostatic type of interaction, assuming that four negative charges on tRNA are neutralized by each spermine molecule. It was estimated that two to three new binding sites for divalent canons are created upon the binding of spermine to tRNATyr.