bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Role of Dihydroorotate Dehydrogenase in Growth Cessation of Ehrlich Ascites Tumor Cells Cultured under Oxygen Deficiency
Authors: LÖFFLER Monika 
Published: March 2005

In pyrimidine biosynthesis the oxidation of dihydroorotate is catalyzed by dihydroorotate dehydrogenase, which is linked to the mitochondrial electron transport chain in vertebrates. In order to elucidate the role of this oxygen‐dependent anabolic process in the arrest of cell proliferation under anaerobic culture conditions, the effect of the antimetabolite dihydro‐5‐azaorotic acid on growth, metabolism and cell cycle distribution of Ehrlich ascites tumor cells was studied and compared with the effect of oxygen‐free culture conditions.Experiments with cell homogenates confirmed that the dehydrogenation of dihydroorotate is blocked by the inhibitor. In intact cells 2 mM dihydro‐5‐azaorotic acid inhibited incorporation of dihydro[6‐14C]orotate into nucleic acids, and no further increase in RNA and DNA content was observed in its presence for several hours. The cells remained viable; glycolylic activity was normal; respiration was reduced; growth cessation occurred within a few hours. DNA histograms obtained by flow cytometry revealed an accumulation of cells in the G1 phase, which was also observed with anaerobically cultured cells. Substitution of cultures with pyrimidine nucleosides completely sustained cell proliferation in the presence of the inhibitor but not in an anoxic atmosphere. It is concluded that in the absence of oxygen cell proliferation may be arrested by inhibition of the dihydroorotate dehydrogenase alone. However, additional impairments of cell metabolism must play an important role.
Substitution in vitro of Lecithin–Cholesterol Acyltransferase
Authors: UTERMANN Gerd , MENZEL Hans‐Jürgen , ADLER Gido , DIEKER Peter , WEBER Wilfried 
Published: March 2005

Lecithin–cholesterol acyltransferase (EC 2.3.1.43) was purified 15000‐fold from human plasma. The active material was homogeneous in different gel electrophoretic systems but separated into three major bands with apparent pI values of 4.28, 4.33 and 4.37 in isoelectrofocusing. The apparent Mr of the enzyme is 67000 ± 2000. An antiserum prepared against the purified enzyme specifically inhibited the activity of lecithin–cholesterol acyltransferase in whole serum.Serum from a patient with familial deficiency of lecithin–cholesterol acyltransferase was substituted in vitro with the highly purified enzyme. The serum from this patient did not contain immunochemically detectable enzyme protein. Substitution of enzyme resulted in the following major changes.

Cholesteryl ester content in serum increased by 36–89 mg/100 ml depending on the experimental conditions. The enzyme‐mediated formation of cholesteryl ester led to an increase of cholesteryl ester content in high‐density and very‐low‐density lipoproteins and in low‐density lipoproteins containing apoprotein‐B. No increase occurred in fractions containing very large flattened structures and the abnormal lipoprotein‐X and in lipoprotein‐E. Incubation of isolated fractions with lecithin‐cholesterol acyltransferase led to significant cholesterol esterification only in high‐density lipoproteins.
The characteristic disc‐shaped rouleaux‐forming high‐density lipoproteins of enzyme‐deficient serum disappeared. Instead a single homogeneous population of high‐density lipoproteins formed. The particles generated were spherical and had the electrophoretic properties, density (1.080 g/ml), diameter (12.5 nm) and apoprotein composition of normal high‐density lipoproteins‐2.
The concentration of spherical particles containing apolipoprotein E (density 1.040–1.080 g/ml) and the lamellar lipoprotein‐X‐like structures in the low‐density lipoprotein fraction were not affected by the enzyme substitution.
A single homogeneous population of spherical lipoprotein‐B particles of 26.5‐nm diameter occurred at density 1.029 g/ml.

The data suggest that the discoidal high‐density lipoproteins are the major site of cholesteryl ester formation that apolipoprotein‐E is not involved in an undirectional transport of newly formed cholesteryl ester from high‐density lipoproteins to other lipoproteins and that lipoprotein‐X and lipoprotein‐E are not preferential substrates for the acyltransferase.
Properties of the Hydrogenase of Megasphaera elsdenii
Authors: VAN DIJK Cees , GRANDE Hans J. , MAYHEW Stephen G. , VEEGER Cees 
Published: March 2005

The catalytic activities of Megasphaera elsdenii hydrogenase are stimulated by salts. The stimulation is due to the anion: the more chaotropic the anion, the greater the effect.Dithionite‐reduced and dye‐oxidised preparations of hydrogenase are inactivated by reaction with oxygen. The inactivation of the reduced enzyme by excess oxygen follows pseudo‐first‐order kinetics; the reaction order for the oxidised enzyme has not been established. The rate of oxygen‐inactivation is decreased by bovine serum albumin.The hydrogen production activity decreases in the presence of dimethylsulphoxide and ethylene glycol. The hydrogen oxidation activity is stimulated by dimethylsulphoxide, and the activity remains linear with time at concentrations up to 50%(v/v). Above 70% dimethylsulphoxide the steady‐state activity of hydrogenase is abolished for both types of activity.The enzyme is more stable in a hydrogen atmosphere than in an argon atmosphere, and the oxidized enzyme is more stable than the reduced enzyme.The enzyme is isolated in the presence of dithionite and it is therefore reduced. When the enzyme is oxidized by treatment with 2,6‐dichloroindophenol or with (bi)sulphite, its activity increases by up to 65%; this activation is not reversed when the enzyme is re‐reduced. The increase in activity is associated with a change of the redox potential of the incubation medium to a less negative value; half of the maximum activation occurs at ‐0.41 V.The electron paramagnetic resonance spectrum of the dithionite‐reduced hydrogenase resembles that of a reduced ferredoxin‐type of spectrum with two 4Fe‐4S clusters. The spectrum of the oxidized enzyme is similar to that of Chromatium high‐potential iron‐sulphur protein. No redox potentials can be ascribed to these spectra since the redox system changes upon freezing to liquid helium temperatures.
The Role of Hydrogen Translocating Shuttles during Ethanol Oxidation in Hepatocytes from Euthyroid and Hyperthyroid Rats
Authors: HENSGENS Lambert A. M. , NIEUWENHUIS Benedictus J. W. M. , VAN DER MEER Roelof , MEIJER Alfred J. 
Published: March 2005

A quantitative study of the contribution of the malate‐aspartate and glycerol‐3‐phosphate cycles to the translocation of reducing equivalents from cytosol to mitochondria during ethanol oxidation has been made in hepatocytes from euthyroid and hyperthyroid rats.1. In hepatocytes from euthyroid rats both cycles have an almost equal capacity and their relative contribution to total hydrogen transport to the mitochondria depends on the conditions chosen.2. In hepatocytes from hyperthyroid rats maximal rates of ethanol oxidation were significantly lower than in hepatocytes from euthyroid rats, even though the capacity of the glycerol‐3‐phosphate cycle was increased. This was due to a decreased activity of alcohol dehydrogenase.
Isomeric Incorporation of the Haem into Monomeric Haemoglobins of Chironomus thummi thummi
Authors: KRÜMPELMANN Dieter , RIBBING Wilfried , RÜTERJANS Heinrich 
Published: March 2005

13C‐NMR studies on 13CO complexes of the components III and IV of the monomeric haemoglobins from the subspecies Chironomus thummi thummi have been carried out in order to confirm the existence of two conformational isomers differing by the isomeric incorporation of the haem group and the extent of the Bohr effect. In addition, the allosteric linkage between the ligand binding site and the Bohr proton binding site in the component IV is described from investigations of various pH‐dependent proton resonances including the C‐2 proton resonances of the titratable histidines. In comparison to the data obtained for the component III it is assumed that in both conformational isomers of the component IV the allosteric linkage between the distal site of the haem and the main Bohr proton donating group is present.From corresponding NMR investigations of the component I an isomeric incorporation of the haem group into this monomeric haemoglobin seems unlikely. Also, any correlation between the ligand binding site and a titratable group of the protein has not been found in this haemoglobin, in agreement with previous results of other laboratories that a Bohr effect in the component I cannot be detected.
Structure and Synthesis of a Lipid‐Containing Bacteriophage Studies on the Structure of the Bacteriophage PM2 Nucleocapsid
Authors: SATAKE Haruhiko , AKUTSU Hideo , KANIA Malgosia , FRANKLIN Richard M. 
Published: March 2005

The nucleocapsid of bacteriophage PM2, prepared according to Schäfer et al. [Eur. J. Biochem. 92, 579–588 (1978)], was studied by biochemical and biophysical methods. It was not possible to isolate the lipid‐free nucleocapsid. More than 95% of the lipids were associated with the nucleocapsid. The asymmetric distribution of phospholipids across the viral membrane was retained in the nucleocapsid since less than 10% of the phosphatidylethanolamine was accessible to the non‐penetrable membrane probe, 2,4,6‐trinitrobenzenesulfonate.Micro‐dissection of the nucleocapsid with thermolysin demonstrated the asymmetric orientation of core proteins across the nucleocapsid membrane. Protein III was embedded deeply in the lipid bilayer and about 20% of the molecule extended to the exterior. Protein IV interacts with PM2 DNA and is partially in the inner leaflet of the bilayer.Small‐angle X‐ray scattering studies on the nucleocapsid enabled us to localize the lipid bilayer structure at radii from 20.5 nm to 24.0 nm.
Isolation and Characterization of Two Molecular Forms of Octopine Dehydrogenase from Pecten maximus L.
Authors: MONNEUSE‐DOUBLET Marie‐Odile , LEFEBURE Francine , OLOMUCKI Anna 
Published: March 2005

Two forms, A and B, of octopine dehydrogenase from Pecten maximus L. striated adductor muscles were separated by ion‐exchange chromatography and purified to homogeneity.Their kinetic properties were similar and, among others, the mnemonical behaviour, previously found for octopine dehydrogenase, was confirmed. Structural features, determined by polyacrylamide gel electrophoresis with and without sodium dodecylsulfate, amino acid composition, immunological tests and heat stability were compared. The only differences between the two forms are their charge and their sensibility to various chemical treatments. Assays of reciprocal conversion of the two forms by oxidation, reduction or deamidation failed. No tissue specificity and no relation to any physiological conditions could be observed. However, a constant ratio A:B = 1:4 was statistically found in crude extracts as well as in the purified enzyme. It therefore seems possible to assume that the two forms of octopine dehydrogenase pre‐exist in living P. maximus, although their genetic origin has to be established.
Structure de la chrysantelline B, nouvelle saponine isolée de Chrysanthellum procumbens Rich
Authors: BECCHI Michel , BRUNETEAU Maud , MICHEL Georges , TROUILLOUD Michel , COMBIER Henri , PONTANIER Henri 
Published: March 2005

Structure of Chrysantellin B, a New Saponin Isolated from Chrysanthellum procumbens RichA new saponin, chrysantellin B, has been found in a tropical plant Chrysanthellum procumbens Rich as a minor companion of chrysantellin A which was previously studied. The structure of chrysantellin B was determined by mass spectrometry, proton and 13C nuclear‐magnetic resonance. The aglycone part is a triterpene: 3β,16α,23‐trihydroxy‐olean‐12‐en‐28‐oic acid or caulophyllogenin. Carbohydrate components are D‐glucose, D‐xylose and L‐rhamnose in a molar ratio 1:2:2, The structure of chrysantellin B was established as 3‐O‐β‐D‐glucopyranosyl[L‐rhamnopyranosyl‐(α1 → 3)‐D‐xylopyranosyl‐(β1 → 4)‐D‐rhamnopyranosyl‐(α1 → 2)‐D‐xylopyranosyl‐(β1 → 28)]caulophyllogenin.
Evidence that Ionization of Zinc‐Bound Water Regulates the Anion‐Binding Capacity of the Coenzyme‐Binding Site in Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , LINDSTRÖM Anders , OLDÉN Bertil , PETTERSSON Gösta 
Published: March 2005

1. The mechanistic and structural origin of the pKa 9.2 dependence of coenzyme association and anion binding to liver alcohol dehydrogenase has been investigated by titrimetric and spectrophotometric binding studies involving ligands (imidazole, 1,10‐phenanthroline and 2,2′‐bipyridine) which combine to the catalytic zinc ion of the enzyme subunit with displacement of zinc‐bound water.2. Imidazole abolishes the pKa 9.2 dependence of NADH binding to the enzyme. The pH dependence of ADP‐ribose and Pt(CN)2‐4 binding is similarly abolished by imidazole, as well as by 1,10‐phenanthroline and 2,2′‐bipyridine. It is concluded from these results that the pKa 9.2 dependence of coenzyme and anion binding most likely derives from ionization of zinc‐bound water.3. Evidence is presented showing that the protonation state of the pKa 9.2 group also regulates ligand binding to the catalytic zinc ion, which lends strong support to the conclusion that this ionizing group can be identified as zinc‐bound water. The pKa, 9.2 dependence of bipyridine binding is abolished by ADP‐ribose and Pt(CN)2‐4, indicating that complex formation at the anion‐binding subsite of the coenzyme‐binding site perturbs the pKa of zinc‐bound water to a value above 10.4. The cooperative interrelationship between ionization of zinc‐bound water and complex formation at the anion‐binding subsite is proposed to be attributable to a competition between the zinc‐bound hydroxyl ion and external anionic ligands for salt bridge formation with the guanidinium group of Arg‐47. This explains why ionization of zinc‐bound water affects the anion‐binding capacity of the enzyme and why complex formation at the anion‐binding subsite affects the pKa of zinc‐bound water.5. The kinetics of complex formation with bipyridine are consistent with a two‐step binding mechanism in which a pKa 9.2 dependent rapid pre‐equilibration between enzyme and ligand is followed by a pH independent rate‐limiting formation of the chromophoric binary complex. Desorption of zinc‐bound water, therefore, is likely to take place in the primary association step rather than in the subsequent rate‐limiting step. This renders the possibility less likely that productive ternary complexes formed during catalysis may contain a zinc‐bound penta‐coordinate water molecule.
Relation of the Antiproliferative Action of Methylglyoxal‐bis(guanylhydrazone) to the Natural Polyamines
Authors: SEPPÄNEN Pauli , ALHONEN‐HONGISTO Leena , JÄNNE Juhani 
Published: March 2005

Enzymic determinations of intracellular concentrations of methylglyoxal‐bis(guanylhydrazone), an anticancer drug which inhibits the synthesis of the polyamines spermidine and spermine, in cultured tumor cells revealed that the drug was remarkably effectively concentrated inside the cell. A concentration gradient across the cell membrane as great as 500–1000‐fold was formed in cells exposed to the drug for 1–2 days. An exposure of cultured Ehrlich ascites carcinoma cells to increasing concentrations of the drug indicated that the cells could tolerate intracellular concentrations up to 1 mM with only slight changes in their proliferation rate. Micromolar concentrations of spermidine or spermine, but not putrescine, effectively blocked the uptake of methylglyoxal‐bis(guanylhydrazone) and reduced the intracellular concentration of the drug below the levels required for growth inhibition.Analysis of cellular polyamine contents in Ehrlich ascites cells exposed to rising concentrations of methylglyoxal‐bis(guanylhydrazone) gave little support to the view that the drug‐induced growth inhibition was solely produced by an intracellular polyamine deprivation.Not only was the uptake of the drug inhibited in the presence of spermidine and spermine, but it was likewise washed out by polyamines from the cells that had been previously exposed to the drug and then transferred into drug‐free medium in the presence of polyamines.For the inhibition of methylglyoxal‐bis(guanylhydrazone) uptake by amines, three or more amino/imino groups were apparently required, since low concentrations of aliphatic diamines were either without any effect (short‐chain diamines) or only marginally prevented (long‐chain diamines) the uptake of the drug. High concentrations of Mg2+ ions, however, markedly inhibited the transport of the drug into Ehrlich ascites tumor cells.
Structure of the 5‐S Ribosomal RNA Gene and Its Adjacent Regions in Torulopsis utilis
Authors: TABATA Satoshi 
Published: March 2005

A DNA segment spanning one repeating unit of ribosomal RNA(rRNA) genes in a yeast strain, Torulopsis utilis, has been cloned on a bacterial plasmid pBR322. The size of the cloned segment was about Mr, = 8.0 × 106. All of the genes for the four species of rRNA were linked on it, and there was a long spacer between the 5‐S and 18‐5 rRNA coding regions.The nucleotide sequences of the regions flanking the 5‐S rRNA gene were determined and compared with those in the corresponding regions of Saccharomyces cerevisiae [Valenzuela, P., Bell, G. I., Masiarz, F. R., DeGennaro, L. J., and Rutter, W. J. (1977) Nature, 267, 641–643; Maxam, A. M., Tizard, R., Skryabin, K. G., and Gilbert, W. (1977) Nature, 267, 643–645]. The sequence of the T. utilis 5‐S rRNA is identical with that of S. cerevisiae except for two residues at positions 18 and 61. However, its upstream region contained a quite different sequence, and a sequence which showed some homology was only found at positions – 21 to – 28. The sequences were d(T‐G‐T‐A‐A‐C‐C‐T) in T. utilis and d(T‐A‐T‐C‐A‐C‐C‐T) in S. cerevisiae. Although the presence of various repeat sequences having the same or opposite directions was noted, these repeats occurred at different positions in the two yeast species.In the downstream region, the common sequence was only seven dT deoxynucleotides, which occurred immediately after the 5‐S rRNA coding sequence. Significant direct and inverted repeat sequences were found in T. utilis, but such repeats are not seen in S. cerevisiae.
The Modification of the Peptidyltransferase Activity of 50‐S Ribosomal Subunits, LiCl‐Split Proteins and L16 Ribosomal Protein by Pyridoxal Phosphate
Authors: BAXTER Ross M. , WHITE Vivienne T. , ZAHID Nasir D. 
Published: March 2005

Pyridoxal phosphate photoinactivates the peptidyltransferase activity of 504 ribosomal subunits, LiCl split proteins and protein L16. Ethyromycin exhibits significant protection.These results, taken together with earlier reports, indicate the involvement of the single histidine of L16 in peptidyltransferase activity. The adjacent association in L16 of histidine and lysine indicates that pyridoxal phosphate should represent a selective inhibitor of peptidyltransferase activity.
Reversible Phosphorylation and Inactivation of Acetyl‐CoA Carboxylase from Lactating Rat Mammary Gland by Cyclic AMP‐Dependent Protein Kinase
Authors: HARDIE D. Grahame , GUY Paul S. 
Published: March 2005

Acetyl‐CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35–70‐fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240000, containing 1.02 ± 0.04 molecules of biotin and 3.1 ± 1.7 molecules of alkali‐labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [γ‐32P]ATP and magnesium ions with the purified catalytic subunit of cyclic‐AMP‐dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat‐stable protein inhibitor of cyclic‐AMP‐dependent protein kinase, and can be reversed by incubation with purified protein phosphatase‐1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near‐stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl‐CoA carboxylase by protein phosphatase‐1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333–338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic‐AMP‐dependent protein kinase or acetyl‐CoA carboxylase kinase‐2.These results suggest that the rapid inactivation of acetyl‐CoA carboxylase, and hence fatty acid synthesis, by adrenaline in adipose tissue, or glucagon in the liver, is due to phosphorylation of the enzyme by cyclic‐AMP‐dependent protein kinase.
Taurocholate Inhibits the Glucocorticoid‐Induced Rise of 3‐Hydroxy‐3‐methylglutaryl‐CoA Reductase in Primary Culture of Hepatocytes
Authors: BARTH Christian A. , HILLMAR Ingrid 
Published: March 2005

The influence of taurocholate, the major bile acid of the rat, on 3‐hydroxy‐3‐methylglutaryl‐CoA reductase [mevalonate: NADP+ oxidoreductase (acylating CoA); EC 1.1.1.34], the regulatory enzyme of cholesterol synthesis, was studied in primary cultures of rat hepatocytes. The basal activity of the enzyme was not altered by adding up to 10 μM taurocholate to the culture medium.On the contrary, 1 nM to 10 μM taurocholate caused a dose‐dependent inhibition of enzyme activity within 6 h if added simultaneously with 10 μM dexamethasone. Because this glucocorticoid causes a cycloheximide‐sensitive rise of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in this system the results are taken as evidence that bile salts inhibit the synthesis of the enzyme. The induction of tyrosine transaminase (L‐tyrosine: 2‐oxoglutarate aminotransferase; EC 2.6.1.5) by dexamethasone was not influenced by taurocholate, which demonstrates that the glucocorticoid sensitivity of the cells was not impaired by the bile salt.It is concluded that there is a direct control of hepatic cholesterol synthesis by bile salts.
Isolation of Polymerization‐Competent Cytoplasmic Actin by Affinity Chromatography on Immobilized DNAse I Using Formamide as Eluant
Authors: ZECHEL Kasper 
Published: March 2005

Formamide dissociates the G‐actin · DNAse‐I complex and is therefore suitable as an alternative to the eluant containing 3 M guanidinium hydrochloride, suggested originally by Lazarides and Lindberg [Proc. Natl Acad. Sci. USA, 71, 4742–4746 (1974)], to elute actin from immobilized DNAse‐I–agarose. Formamide provides the advantage of being a much weaker denaturant than guanidinium hydrochloride and being a nonionic substance. In the concentration necessary for the dissociation of the G‐actin · DNAse‐I complex (approximately 10 M) formamide denatures actin only slowly (half‐time approximately 150 min at 2°C) and thus allows the recovery of a large fraction of actin in a polymerization‐competent form from the affinity column.Based on these findings a rapid two‐step procedure for the isolation of non‐muscle G‐actin from cultured cells is described. The actin is obtained in high yield and purity (greater than 90%) and can readily polymerize to F‐actin.