bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nucleolar DNA‐Dependent RNA Polymerase from Rat Liver
Authors: MATSUI Takashi , ONISHI Toshio , MURAMATSU Masami 
Published: March 2005

RNA polymerase I (or A) was extracted from nuclear, nucleolar and nucleoplasmic fractions, and resolved into IA and IB forms on a phosphocellulose column. During the course of cyclo‐heximide treatment, the activity of RNA polymerase IB decreased in the nucleoli with concomitant increase in the nucleoplasmic fraction, suggesting strongly that cycloheximide induced specific leakage of IB enzyme from the nucleolus. The activity of IA enzyme did not change in the nucleoli.When nucleoli were incubated in the presence of actinomycin D, all the IA enzyme activity and approximately 30% of the total IB enzyme activity were released in the incubation medium, whereas 70% of IB activity remained associated with the nucleolar pellet where no IA activity was detected. The enzyme which was released into the incubation medium was tentatively designated as free or unbound RNA polymerase I and that which was associated with the nucleolar pellet as template‐bound enzyme. During the treatment with cycloheximide, the activity of bound enzyme, which contained exclusively IB form, decreased rapidly, with kinetics almost identical to that of nucleolar RNA synthesis in vivo. The activity of free enzyme did not change appreciably.At 2 h after partial hepatectomy, IB enzyme activity in the free RNA polymerase fraction increased to almost twice the control, while the bound enzyme activity did not increase appreciably until 4 h of regeneration. Enhancement of nucleolar RNA synthesis in vivo was not apparent at 2 h but became significant by 4 h after partial hepatectomy.These results strongly suggest that (a) the above‐mentioned procedure is actually fractionating RNA polymerase I into free and bound forms, (b) RNA polymerase IB is the transcriptionally active form in vivo, (c) RNA polymerase IB exists in excess in the nucleoli, but the amount of bound IB molecules, which are engaged in transcription in vivo, must be determined by some other factor(s) than the mere concentration of IB enzyme in the nucleolus, and (d) IA form is not an artifact of isolation but is always present in vivo at a certain amount, although the exact nature of this molecule is not known at present.
Excretion of Taurocholate from Isolated Hepatocytes
Authors: SCHWARZ Leslie R. , SCHWENK Michael , PFAFF Erich , GREIM Helmut 
Published: March 2005

Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux.Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonyl‐cyanide‐m‐chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 μM. Ouabain increased the rate of efflux.The observations indicate that secretory functions are maintained in isolated liver cells.
Isolation and Characterization of Hydrophobic Proteins (H Proteins) in the Membrane Fraction of Bacillus subtilis
Authors: KUSAKA Iwao , HAYAKAWA Kou , KANAI Keiko , FUKUI Sakuzo 
Published: March 2005

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155–1.158 g/cm3); medium band (1.181–1.183 g/cm3); heavy band (1.21–1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5–8 nm in diameter. We named this H‐protein.H‐protein formed trilamellar unit membrane structure when combined with lipid. In pulse‐labeling and pulse‐chase experiments with radioactive leucine, it was found that H‐protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H‐protein was shifted into light and medium band of the membranes during the chase.Cytochromes were not found in H‐protein. However, when H‐protein was incubated with haem α and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H‐protein and form cytochromes a and b. Cytochromes were also formed in H‐protein which were isolated from the cells grown in the presence of haemin (haemin‐grown H protein).Succinate dehydrogenase activity was increased about 4‐fold by combining H‐protein or haemin‐grown H‐protein with lipid. H‐protein had no cytochrome oxidase activity; however, haemin‐grown H protein was found to have some of the activity and this was increased about 4‐fold by combining the protein with lipid. Haemin‐grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin‐grown H protein and was activated about 9‐fold in constituted reaction systems. Vesicles formed by haemin‐grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+‐diffusion via valinomycin.These results would indicate that H‐protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.
An Assessment of Some of the Methods Available for the Determination of Molecular Weights of Proteins as Applied to Aspartate Aminotransferase from Pig Heart
Authors: BANKS Barbara E. C. , DOONAN Shawn , FLOGEL Mirna , PORTER Patricia B. , VERNON Charles A. , WALKER John M. , CROUCH Thomas H. , HALSEY John F. , CHIANCONE Emilia , FASELLA Paolo 
Published: March 2005

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g‐1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g‐1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300).A new non‐empirical method based on quantitative N‐terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity.Some of the methods available for the determination of molecular weights and subunit compositions of proteins are discussed.
The Dissociation of Flavin Coenzymes from Trypsin‐Solubilized NADPH/Cytochrome c (P‐450) Reductase of Pig‐Liver Microsomes
Authors: TROUT Gordon E. 
Published: March 2005

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P‐450) reductase.In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first‐order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4–3.8 (0.02 M acetate buffer) where the rate constants increased 80‐fold.Modification of the enzyme by treatment with p‐chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first‐order dissociations. It was concluded that p‐chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates.Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve‐fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein.
Magnetic Studies on the Ferri‐haem Undecapeptide of Cytochrome c
Authors: JEHANLI Ahmed M. T. , STOTTER David A. , WILSON Michael T. 
Published: March 2005

The pH‐dependence of the magnetic moment of a ferri‐haem undecapeptide, produced by peptic digestion of cytochrome c, has been measured in aqueous solution using a nuclear magnetic resonance method.Below pH 3 the magnetic moment is consistent with the presence of hydroxo‐bridged dimers of high‐spin iron(III). Above pH 7 the iron(III) is low‐spin, the spin crossover conforming to a simple titration curve with pK 6.3 (n= 1). This transition is discussed with reference to spectrophotometric studies of ligand binding at the haem.
Rhodanese‐Mediated Sulfur Transfer to Succinate Dehydrogenase
Authors: BONOMI Franco , PAGANI Silvia , CERLETTI Paolo , CANNELLA Carlo 
Published: March 2005

The interaction of the sulfurtransferase rhodanese (EC 2.8.1.1) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied.Succinate dehydrogenase incorporates the sulfane sulfur of [35S]rhodanese and, in the presence of unlabelled rhodanese, also incorporates that of [35S]thiosulfate. Rhodanese releases most of its transferable sulfur and is re‐loaded in the presence of thiosulfate. Rhodanese undergoes similar modifications with yeast alcohol dehydrogenase but this latter does not bind 35S in amounts comparable to those incorporated in succinate dehydrogenase: nearly all the 35S released by [35S]rhodanese is with low‐molecular‐weight compounds. Bovine serum albumin also binds very little sulfur and [35S]rhodanese present in the reaction mixture does not discharge its radioactive sulfur nor does it take up sulfur from thiosulfate.Sulfur release from rhodanese appears to depend on the presence of – SH groups in the acceptor protein.Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron‐sulfur absorption of the flavoprotein.The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation.The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.
Ribitol‐Containing Lipopolysaccharides from Proteus mirabilis and Their Serological Relationship
Authors: GMEINER Jobst , MAYER Hubert , FROMME Inge , KOTELKO Krystyna , ZYCH Krystyna 
Published: March 2005

Ribitol phosphate was recently identified as a constituent of lipopolysaccharides obtained from Proteus mirabilis strain D52 giving 1:4‐anhydroribitol during acid hydrolysis (Gmeiner, 1975). Two other Proteus mirabilis strains belonging to serogroups O16 and O33 were shown previously to contain an unknown compound X as lipopolysaccharide constituent (Kotelko et al., 1975).In this report the identification of compound X as 1:4‐anhydroribotol by gas‐liquid chromatography, mass spectrometry and mass fragmentography is described.Serological investigations using passive hemagglutination, hemagglutination inhibition and semiquantitative precipitin reactions indicate strongly that ribitol plays a role in the serological specificity of the respective lipopolysaccharides.
Lignoceric Acid Biosynthesis in the Developing Brain. Activities of Mitochondria1 Acetyl‐CoA‐Dependent Synthesis and Microsomal Malonyl‐CoA Chain‐Elongating System in Relation to Myelination
Authors: BOURRE Jean‐Marie , PATURNEAU‐JOUAS Marion Y. , DAUDU Odile L. , BAUMANN Nicole A. 
Published: March 2005

Age‐related changes in the activities of microsomal and mitochondrial elongating systems have been determined in mouse brain from birth to maturity.In microsomes, the components necessary for behenyl‐CoA (docosanoyl‐CoA) elongation have been found to be NADPH and malonyl‐CoA. In mitochondria, both NADH and NADPH are used and acetyl‐CoA is the only donor of two‐carbon‐atoms unit. The synthesised fatty acids were identified by thin‐layer and gas chromatography. The specific activity is higher in microsomes than in mitochondria.In microsomes, the specific activity for malonyl‐CoA incorporation reached a maximum at 15–20 days of age; this peak was not obtained in the Quaking and Jimpy mutants. The increase in enzyme activity (specific activity and total activity per brain) paralleled the myelin deposition. The activity of the mitochondrial system increases regularly during development: it is not correlated to myelination and it is not affected in the Quaking mutant. The interplay between microsomal and mitochondrial elongation systems is studied.
Assymetry of the myo‐Inositol Transport System in Klebsiella aerogenes
Authors: DESHUSSES Jacques , REBER Guido 
Published: March 2005

In Klebsiella aerogenes the influx of myo‐inositol proceeds during the steady state at a rate equal to that of efflux.
The kinetic parameters of influx during the steady state are similar to those observed at the initial time of uptake.
Efflux and influx processes share the same transport system.
The ability of other cyclitols to chase the accumulated scyllo‐inositol is dependent on their affinity for the transport system.
A counter transport can be observed in preloaded cells only in poisoned cells and under anaerobiosis.
The efflux process is not temperature‐dependent.
The KT of influx in poisoned cells is larger than that in normal cells.
Although no saturation kinetics of efflux could be observed, it can be inferred that the KT of efflux is at least 50‐fold larger than the KT of influx;
The results suggest that in the transport of myo‐inositol by K. aerogenes energy coupling enhances the affinity of the carrier for the substrate at the outer side of the cytoplasmic membrane by lowering the KT.
Inequivalent Conformational Response of Chironomus Hemoglobins to Ligation with O2 and CO.
Authors: WOLLMER Axel , STEFFENS Guy , BUSE Gerhard 
Published: March 2005

For the monomeric hemoglobins III and IV and the dimeric hemoglobins of Chironomus the tryptophan circular dichroism reports different conformational response to binding of different ligands. The fine structure bands at 292 nm are most strongly developed in the unliganded state and considerably reduced in the oxy form. The spectrum of the CO derivative is intermediate but clearly more deoxy‐like. This finding correlates well with the corresponding Bohr effect magnitudes determined by measuring proton release upon ligation.Infrared difference spectroscopy on O2versus CO derivatives in aqueous solution shows a normal O2 stretching band position at 1107 cm−1 characteristic of asymmetric oxygen binding. The CO stretching band, however, is consistently blue‐shifted by 11–13 cm−1 from the 1951 cm−1 position observed with mammalian hemoglobins, indicating reduced CO binding strength.Structural factors relevant to an explanation of the observed phenomena are discussed.
CO2‐Mediated Control of Fatty Acid Metabolism in Isolated Hamster Brown‐Fat Cells during Norepinephrine Stimulation
Authors: PETTERSSON Bertil 
Published: March 2005

Addition of norepinephrine to isolated hamster brown‐fat cells suspended in Krebs‐Ringer phosphate buffer induces a pronounced, but temporary, increase in respiratory rate.
If Krebs‐Ringer phosphate buffer is bubbled with CO2 prior to the addition of cells and norepinephrine, the respiratory capacity of the cells is further potentrated and most important, the respiration is maintained at a high rate until the medium becomes depleted of oxygen.
This respiratory pattern cannot be obtained in CO2‐bubbled Krebs‐Ringer bicarbonate buffer.
The results indicate that CO2 has a regulatory effect on fatty acid metabolism in isolated hamster brown‐fat cells.
Organization of Sequences in Avian Globin mRNA
Authors: CRAWFORD Robert John , SCOTT Andrew Charles , WELLS Julian Richard E. 
Published: March 2005

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 650 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions.

45–50 of these untranslated nucleotides are present as poly(A) at the 3′ terminus.
The 3′ untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lengths of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion.
It is probable that there is greater evolutionary divergence in the 3′ untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules.

The combined data is used to formulate a regional map of chicken globin mRNA.
Mitochondrial and Cytosolic NADPH Systems and Isocitrate Dehydrogenase Indicator Metabolites during Ureogenesis from Ammonia in Isolated Rat Hepatocytes
Authors: SIES Helmut , AKERBOOM Theodorus P. M. , TAGER Joseph M. 
Published: March 2005

Citrate, isocitrate and 2‐oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions thereof, obtained by the digitonin and silicone oil fractionation technique.
Calculated from isocitrate/2‐oxoglutarate ratios (“indicator metabolite method”), the redox potential of mitochondrial free NADPH is – 402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, – 392 mV.
Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, as demonstrated by spectrophoto‐metry of the dihydro band and by the changes in the isocitrate/2‐oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reversed.
It is concluded that during ureogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP‐linked as opposed to the NAD‐linked enzyme; isocitrate concentration under these conditions is less than 10 μM, below the Km (isocitrate) of the NAD‐linked enzyme but in the range of that for the NADP‐linked enzyme.
Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2‐oxoglutarate.
These results and data in the literature on enzyme activity are in agreement with the assumption of near‐equilibrium of NADP‐dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to ‐391 mV or possibly even higher if there exists an indicator error under this condition.
Inhibition of Translation in Eukaryotic Systems by Harringtonine
Authors: FRESNO Manuel , JIMÉNEZ Antonio , VÁZQUEZ David 
Published: March 2005

The Cephalotaxus alkaloids harringtonine, homoharringtonine and isoharringtonine inhibit protein synthesis in eukaryotic cells. The alkaloids do not inhibit, in model systems, any of the steps of the initiation process but block poly(U)‐directed polyphenylalanine synthesis as well as peptide bond formation in the fragment reaction assay, the sparsomycin‐induced binding of (C)U‐A‐C‐C‐A‐[3H]Leu‐Ac, and the enzymic and the non‐enzymic binding of Phe‐tRNA to ribosomes. These results suggest that the Cephalotaxus alkaloids inhibit the elongation phase of translation by preventing substrate binding to the acceptor site on the 60‐S ribosome subunit and therefore block aminoacyl‐tRNA binding and peptide bond formation.However, the Cephalotaxus alkaloids do not inhibit polypeptide synthesis and peptidyl‐[3H]puromycin formation in polysomes. Furthermore, these alkaloids strongly inhibit [14C]trichodermin binding to free ribosomes but hardly affect the interaction of the antibiotic with yeast polysomes. These results clearly suggest that the Cephalotaxus alkaloids cannot interact with polysomes and therefore only inhibit the initial cycles of elongation. This explains the polysome run off that has been observed by some workers in the presence of harringtonine.
The Primary Structure of Yeast Alcohol Dehydrogenase
Authors: JÖRNVALL Hans 
Published: March 2005

Eight different types of peptide mixtures from [14C]carboxymethylated yeast alcohol dehydrogenase were obtained using trypsin with or without prior maleylation of the substrate, chymotrypsin, pepsin, microbial proteases or CNBr. Each mixture was fractionated by exclusion chromatography and peptides were further purified on paper. From results of analyses of all fragments it seems possible to deduce a primary structure of 347 unique residues in three segments. Together, the segments can account for the whole protein monomer with the exception of a small connecting region. Many unfavourable structures complicated the determination and made single sequence conclusions tentative, but known data are consistent and for most segments of the monomer results are abundant.Several microheterogeneities in the protein are indicated and one apparent amino acid exchange is characterized, suggesting that different types of subunits occur. This may probably be correlated with genetic polymorphism in yeast. Multiple desamidations are also characterized and a few of these affect particularly labile structures.Many residues are unevenly distributed and unexpected patterns are shown. Elements of repetitive sequences occur, reducing the uniqueness of structures. Hydrophobic segments are found, and the uncharacterized region is, at least in some subunits, in a core‐like tryptic segment. These and other aspects of the structure may explain some properties of the monomer, and form the background for evolutionary, structural and functional correlations with releated enzymes.