bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fractionation of Rat‐Liver‐Chromatin Nonhistone Proteins into Two Groups with Different Metabolic Rates
Authors: RUSSEV George , ANACHKOVA Boika , TSANEV Roumen 
Published: March 2005

In the pH interval 10.5–11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13.Dodecylsulfate‐polyacrylamide gel electrophoresis revealed that the majority of the high‐molecular‐weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40000–60000 remained in the alkali‐resistant group. l‐[14C]Leucine pulse‐labelling experiments showed that the specific radioactivity of the alkalilabile nonhistone proteins was 2–3 times higher than that of the alkali‐resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver.In the course of a 21‐day chase the specific radioactivity of the alkali‐labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali‐resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali‐resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase.A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali‐resistant and is conserved in chromatin like histones.
On the Dissociation and Association of Escherichia coli Ribosomes
Authors: WALTERS Hans , SMITS Willem , VAN KESSEL Jan , VAN OS Gerard 
Published: March 2005

The dissociation and association behaviour of 70‐S ribosomes of Escherichia coli has been studied. It has been shown that the dissociation‐association reaction can be both a real dynamic equilibrium and a non‐equilibrium reaction, dependent upon the ionic conditions of the solvent. At relatively high ionic strength (I= 0.15 M or more) the dissociation‐association reaction is an equilibrium reaction, whereas at lower ionic strength (I= 0.1 M or less) there is no dynamic equilibrium between 70‐S ribosomes and its subunits. In the latter case a hysteresis in the dissociation‐association reaction is observed.Whether there is a dynamic equilibrium or not can be demonstrated by a single centrifugation experiment, using the analytical ultracentrifuge.
Polynucleotide Kinase from Rat‐Liver Nuclei
Authors: TERAOKA Hirobumi , MIZUTA Kazutaka , SATO Fumiyasu , SHIMOYACHI Mari , TSUKADA Kinji 
Published: March 2005

A polynucleotide kinase, which catalyzes the phosphorylation of 5′‐hydroxyl ends of deoxyribo‐nucleic acid in the presence of adenosine triphosphate, has been purified 260‐fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei.The purified enzyme has a pH optimum of 5.5. The enzyme is reversibly inhibited by p‐chloro‐mercuribenzoate. The S0.5 value (ligand concentration required for a half‐maximal activity) for ATP is 2.5 μM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkably inhibits the activity with I0.5 value (ligand concentration required for a half‐maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM.An apparent molecular weight of the purified enzyme is estimated to be 8 × 104 by gel filtration on a column of Sephadex G‐150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.
Ultrastructural Organization and Biochemical Characterization of Chromatin · RNA · Protein Complexes Isolated from Mammalian Cell Nuclei
Authors: BACHELLERIE Jean‐Pierre , PUVION Edmond , ZALTA Jean‐Pierre 
Published: March 2005

Extranucleolar elements were isolated from mammalian cell nuclei and characterized by correlative biochemical‐ultrastructural studies. These complex structures were shown to retain a supra‐particle arrangement closely resembling the organizaton in situ of definite nuclear areas. In particular the morphological RNA · protein species, namely perichromatin fibrils and granules, were detected in association with characteristic regions of chromatin. By autoradiographic experiments, we were able to show that perichromatin fibrils represent the morphological state of newly formed heterogeneous nuclear RNA (hnRNA).Chromatin · RNA · protein complexes were further fractionated by means of a treatment dissociating the chromatin component. A minor part of DNA, possibly involved in the biosynthesis and the processing of hnRNA, remains linked to the resulting RNA · protein network.
Phenol Hydroxylase from Yeast
Authors: NEUJAHR Halina Y. , GAAL Andràs 
Published: March 2005

Thiol groups in phenol hydroxylase were measured using two different –SH reagents and amino acid analysis. Stepwise blocking of the –SH groups was correlated with enzyme activity and FAD content.The results indicate that the enzyme contains 16 –SH groups per molecule of Mr 1.48 × 105. At least four –SH groups are not accessible without the use of a denaturing agent. There is seemingly no disulphide bridge.On the whole, the reactivity towards p‐hydroxymercuribenzoate is much greater than towards 5,5′‐dithio‐bis(2‐nitrobenzoic acid). The two reagents seem to have a different specificity with respect to which –SH groups they attack. Either reagent dislocates FAD from the holoenzyme, leaving a characteristic mercaptide derivative of the apoenzyme. Such derivatives were used to prepare the apoenzyme. The –SH groups in the apoenzyme are much more reactive towards 5,5′‐dithiobis(2‐nitrobenzoic acid) than the –SH groups in the holoenzyme.The stoichiometry of the reaction with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) indicates that at least 8 –SH groups are located in spatially close pairs. The most reactive pair of all does not appear to be of importance for enzyme activity. The two subsequent –SH pairs are essential for enzyme activity and are involved in FAD attachment.The reactivity of the –SH groups decreases dramatically in the presence of substrate, even at substrate concentrations equivalent to the level of the catalytic sites.The isolated apoenzyme has a tendency to aggregate. A large proportion of –SH groups in such aggregate(s) is buried, especially when EDTA is not used throughout the preparation of the apoenzyme. The aggregates are enzymically inactive.
The Amino‐Acid Sequence of Isoinhibitor K from Snails (Helix pomatia)
Authors: TSCHESCHE Harald , DIETL Thomas 
Published: March 2005

Performic‐acid‐oxidized isoinhibitor K of snails (Helix pomatia) was subjected to argininedirected tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg‐3‐Pro‐4 bond were purified to homogeneity. Four arginine peptides and the C‐terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular‐ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide <Glu‐Gly revealed that the N‐terminus was blocked by pyroglutamic acid.The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin‐kallikrein® inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.
Patulin Biosynthesis: The Metabolism of m‐Hydroxybenzyl Alcohol and m‐Hydroxybenzaldehyde by Particulate Preparations from Penicillium patulum
Authors: MURPHY Gillian , LYNEN Feodor 
Published: March 2005

The ring hydroxylation of m‐hydroxybenzyl alcohol to gentisyl alcohol by a particulate preparation from Penicillium patulum has been characterised. The activity was shown to be closely associated with, but not necessarily identical to, m‐cresol 2‐hydroxylase activity of the 105 000 x g microsomal fraction. As with both the m‐cresol hydroxylases of this system, m‐hydroxybenzyl alcohol hydroxylase requires oxygen and NADPH for activity. A Km value for m‐hydroxybenzyl alcohol of 15 μM was measured. Inhibition of the hydroxylase activity and its reversal by light, as well as the action of cytochrome c, KCN and other effectors suggested a mixed‐function oxidase reaction of the cytochrome P‐450, NADPH‐cytochrome reductase type. m‐Hydroxybenzaldehyde was not ring hydroxylated by any preparation from P. patulum. Apart from the previously described conversion to m‐hydroxybenzyl alcohol by a predominantly soluble dehydrogenase, m‐hydroxybenzaldehyde was metabolized to m‐hydroxybenzoic acid by a particulate fraction. This activity required NADPH.It was concluded that the main biosynthetic pathway to patulin must be through m‐hydroxybenzyl alcohol, gentisyl alcohol and gentisaldehyde.
Tightness and Orientation of Vesicles from Guinea‐Pig Kidney Estimated from Reactions of Adenosine Triphosphatase Dependent on Sodium and Potassium Ions
Authors: WALTER Horst 
Published: March 2005

In order to study the “sidedness” of the ligands of the Na+, K+‐ATPase in the phosphorylation from [32P] ATP, tight vesicles were prepared from guinea pig kidney and partially purified by a two‐stage sucrose and Ficoll gradient centrifugation procedure. These vesicles were derived presumably from plasma membrane fragments resealed after the initial disruption of the cells during homogenization. Tightness of the vesicles was estimated according to activation by the nonionic detergent, Triton X‐100. Treatment with Triton X‐100 increased both the activity of the Na+, K+‐ATPase and its Na+‐dependent phosphorylation from [32P] ATP at least three‐fold. Activation of both functions also appeared when the vesicles were shocked osmotically. These results suggest that the preparation contains a major population of tight normal vesicles (∼ 75%) in which the phosphorylation site faces faces the intravesicular solution.In the response to ouabain breakdown of the phosphoenzyme was inhibited in vesicles treated with Triton X‐100 but not in intact ones as if ouabain could not get to its binding site. Correspondingly in phosphorylation from ATP pretreatment with ouabain in the presence of inorganic phosphate produced less inhibition in intact vesicles than in those disrupted with Triton X‐100 beforehand.These data suggest the presence of an everted vesicle fraction in the preparation (∼ 20%). Apparently only a small fraction of the vesicles was leaky.In the everted vesicles the action of K+ on the phosphoenzyme was slow. In order to accelerate the dephosphorylation in intact vesicles as effectively as in disrupted ones, K+ had to be added before the start of phosphorylation. This supports the view that K+ was acting from the side of the membrane opposite to that where the γ‐phosphoryl group was accepted from ATP.
The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii
Authors: BRESTERS Tjarda W. , DE ABREU Ronney A. , DE KOK Arie , VEEGER Cees , VISSER Jaap 
Published: March 2005

The pyruvate dehydrogenase complex from Azotobacter vinelandii was isolated in a five‐step procedure. The minimum molecular weight of the pure complex is 600000, as based on an FAD content of 1.6 nmol · mg protein−1. The molecular weight is 1.0–1.2 × 106, indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr 89000) and lipoamide dehydrogenase (Mr monomer 56000) two active transacetylase isoenzymes are present with molecular weight on the gel 82000 and 59000 but probably actually lower.The pure complex has a specific activity of the pyruvate‐NAD+ reductase (overall) reaction of 10 units · mg protein−1 at 25°C. The partial reactions have the following specific activities in units · mg protein−1 at 25°C under standard conditions: pyruvate‐K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor.Under aerobic conditions pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 μmol O2· min−1· mg−1, Km (pyruvate) 0.3 mM. The kinetics of this reaction shows a linear 1/velocity – 1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity ist stimulated by AMP and sulphate and is inhibited by acetyl‐CoA.The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.
Yeast Hexokinase A
Authors: ROSSI Antonio , MENEZES Luiz C. , PUDLES Julio 
Published: March 2005

Yeast hexokinase A (ATP: d‐hexose 6‐phosphotransferase, EC 2.7.1.1) dissociates into its sub‐units upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the same order as those found for the native enzyme.Of the 37 amino groups present per enzyme subunit, 2–3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50 % loss of the enzyme activity may be related to the dissociation of the dimer into monomers.Both native enzyme and the succinylated subunits have the same pH‐dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form.The succinylated subunit presents ‘negative co‐operativity’ with respect to ATP at slightly acid pH; however, the burst‐type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit.
Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin
Authors: ROCCA‐SERRA José , MILILI Michéle , FOUGEREAU Michel 
Published: March 2005

The complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,x) has been determined, thus completing the sequence determination of the entire heavy chain.The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic‐specific regions.
Properties of Immobilized Flavodoxin from Peptostreptococcus elsdenii
Authors: MAYHEW Stephen G. , STRATING Marijke J. J. 
Published: March 2005

The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr‐activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin‐free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively.Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (± 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N‐10 side‐chain of 5 carbon atoms and a C‐5′ phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12–15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months.
Reversible, Covalent Immobilization of Enzymes by Thiol‐Disulphide Interchange
Authors: CARLSSON Jan , AXÉN Rolf , UNGE Torsten 
Published: March 2005

α‐Amylase and α‐chymotrypsin have been immobilized by covalent attachment to mercaptohydroxypropyl ether agarose gel. The technique involves two steps: (a) thiolation of the enzymes by methyl 3‐mercaptopropioimidate, (b) coupling of the thiolated enzymes to a mixed disulphide derivative of agarose obtained by reacting mercaptohydroxypropyl ether agarose with 2,2′‐dipyridyl disulphide.
The immobilization technique can be performed so that most of the inherent activity of the enzymes is conserved. However, diffusion limitations and steric factors prevent full manifestation of the immobilized activities.
Immobilized α‐amylase was used in a packed‐bed reactor for the continuous hydrolysis of starch. When the enzymically active gel had lost its activity it could be regenerated in situ by reductive uncoupling of the inactive protein and attachment of a new portion of thiolated α‐amylase.
On the Mechanism of Protein‐Synthesis Inhibition by Abrin and Ricin
Authors: BENSON Stephen , OLSNES Sjur , PIHL Alexander , SKORVE Jon , ABRAHAM Abraham K. 
Published: March 2005

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell‐free systems from rabbit reticulocytes and Krebs II ascites cells.In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl‐tRNA, demonstrating that the toxins act at some point after the charging of tRNA.Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti‐toxins were added. Recombination experiments between toxin‐treated ribosomes and untreated supernatant factors and vice versa showed that the toxin‐treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis.Recombination experiments between toxin‐treated isolated 40‐S subunits and untreated 60‐S subunits and vice versa showed that only when the 60‐S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited.Both the EF‐1‐catalyzed and the EF‐2‐catalyzed ability of the ribosomes to hydrolyze [γ‐32P]GTP was inhibited by abrin and ricin.An 8‐S complex released from the 60‐S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8‐S complex were inhibited by abrin and ricin.The present data indicate that there is a common site on the 60‐S subunits for EF‐1‐ and EF‐2‐stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.
Extracellular Enzyme System Utilized by the Fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the Breakdown of Cellulose
Authors: STREAMER Margaret , ERIKSSON Karl‐Erik , PETTERSSON Bert 
Published: March 2005

A strong synergistic response was observed between the five endo‐1,4‐β‐glucanases and the exo‐1,4‐β‐glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de‐waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid‐swollen cellulose, was used.If de‐waxed cotton was pretreated with endo‐1,4‐β‐glucanases, the exo‐1,4‐β‐glucanase enzyme released much more degradation products than from an untreated cotton. This strongly supports the theory that the endo‐1,4‐β‐glucanases, acting randomly over the cellulose chain, go in first and open up chain ends where the exo‐enzyme can act.In an earlier paper [Eriksson, K.‐E. & Pettersson, B. (1975) Eur. J. Biochem. 51, 193–206] physico‐chemical differences between the endo‐1,4‐β‐glucanases were demonstrated. Functional differences have now been found. The five endo‐enzymes give rise to different degradation products from Avicel, show differences in preferred mode of attack on cellodextrins, and exhibit pronounced differences in synthetic activity.The exo‐1,4‐β‐glucanase released its products in the α‐configuration.A method for the separation of cellodextrins on a Biol‐Sil A column is also described.
Factors Affecting the Molecular Structure and the Agglutinating Ability of Concanavalin A and Other Lectins
Authors: HUET Marianne 
Published: March 2005

Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature.It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer‐tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4° up to 37°C).It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin‐A‐induced agglutination was previously found to be temperature‐sensitive.These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and not only to modification of cell surface properties.