bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and Quantification of the Androgen and Glucocorticoid Receptors in Cytosol from Rat Skeletal Muscle
Authors: SNOCHOWSKI Marek , DAHLBERG Erik , GUSTAFSSON Jan‐Åke 
Published: March 2005

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9α‐fluoro‐11β, 17α, 21‐trihydroxy‐16α‐methyl‐1, 4‐pregnadiene‐3,20‐dione) and [3H]methyltrienolone (17β‐hydroxy17‐α‐methyl‐4,9,11‐estratrien‐3‐one) to cytosol from rat skeletal muscle was studied using dextrancoated charcoal to separate unbound and receptor‐bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid‐receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0, 15 or 23° C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were –41 and –49 kJ mol‐1 under equilibrium conditions at 0° C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively.Both steroid‐receptor complexes were retained on DNA‐cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA‐cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid‐receptor complexes were excluded from a column of Sephadex G‐150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 0.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.
Opposite Effects of Thiocyanate on Tyrosine Iodination and Thyroid Hormone Synthesis
Authors: VIRION Alain , DEME Danièle , POMMIER Jacques , NUNEZ Jacques 
Published: March 2005

The effect of a pseudohalide, SCN−, an anion with the same molecular size as iodide, was studied on two reactions: thyroglobulin iodination and thyroid hormone synthesis (coupling reaction) catalyzed by peroxidases. The coupling reaction was studied separately from the iodination reaction by using labelled thyroglobulin samples previously iodinated but containing little or no hormones.

SCN− inhibits iodide oxidation (I−→ I2) whatever the enzyme, thyroid, lactoperoxidase or horseradish peroxidase. The amount of SCN− required to completely inhibit this reaction varies depending on the enzyme. Similarly tyrosine iodination is inhibited by SCN− with large variations, depending on the peroxidase, in the concentration of this anion required for inhibition.
In contrast SCN− stimulates the coupling reaction: (a) this effect is seen with the thyroid and lactoperoxidases but not with horseradish peroxidase; (b) the concentration of SCN− required for half‐maximal stimulation of the coupling reaction is much lower (0.5–1 μM) than that required for the inhibition of iodide oxidation (60–80 μM); (c) CIO−4, an anion with the same molecular size as SCN− and I−, has no effect on the coupling reaction; (d) this stimulatory effect of SCN− does not depend on a modification of the thyroglobulin molecule since it is not seen with horseradish peroxidase or in purely chemical coupling conditions.
The stimulatory effect of SCN− is therefore seen as resulting from the binding of this anion to a limited number of high‐affinity sites present at the surface of both thyroid and lactoperoxidases. The inhibitory effect depends, in contrast, on the binding of SCN− to the substrate site with lower affinities. Since iodide also behaves both as a substrate for the iodination reaction and as a stimulatory ligand for the coupling reaction, these data provide further support in favour of the existence of an enzyme‐iodide (or SCN−) complex with catalytic properties different from those of the native peroxidase.
Conformational Characteristics of the Trinucleoside Diphosphate dApdApdA and Its Constituents from Nuclear Magnetic Resonance and Circular Dichroism Studies
Authors: OLSTHOORN Cornelis S. M. , BOSTELAAR Lein J. , VAN BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

Proton NMR studies at 360 MHz were carried out on the trinucleoside diphosphate dApdApdA, the dinucleoside monophosphate dApdA and the methylphosphate esters of the monomers pdA, pdAp and dAp. The compounds were also investigated by means of circular dichroism at varying temperatures. Complete NMR spectral assignments are given. The thermodynamics of stacking derived from the circular dichroic spectra was used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the pure stacked states of the dimer and the trimer.The data were interpreted in terms of the N and S deoxyribose pseudorotational ranges [Altona, C. and Sundaralingam, M. (1972) J. Am. Chem. Soc. 94, 8205–8212] and it is shown that the right‐handed single helix of the trimer occurs in two distinct forms: the major one corresponds to a B‐DNA‐like structure, S‐S‐S; the minor one (30%) has the sugar rings in a hitherto unsuspected S‐S‐N conformational sequence. The dimer behaves quite similarly, a mixture of 70% S‐S and 30% S‐N stacks being indicated. The detailed geometry of the S‐type sugar rings is not invariable but appears to undergo a slight shift when another base stacks on top of a given nucleotide unit. The backbone angles of the fully stacked B‐DNA‐like single helix in solution are (starting the notation at P) β= 187°, γ= 50°, δ= 138°, ɛ= 186°.
Changes in Polygalacturonase Isoenzymes during the ‘Ripening’ of Normal and Mutant Tomato Fruit
Authors: TUCKER Gregory A. , ROBERTSON Neil G. , GRIERSON Donald 
Published: March 2005

Polygalacturonase activity is not detectable in mature green tomato fruits but appears as fruits begin to change colour and continues to increase during the ripening period. There is a sequential appearance of two isoenzymes, polygalacturonase 1 and 2, during ripening. These isoenzymes have been purified and their properties compared. Polygalacturonase 1 has a Mr of 100000, is 50% inactivated at 78° C and has a density of 1.343 g cm−3 in caesium chloride. Polygalacturonase 2 has a Mr of 42000, is 50% inactivated at 57° C and has a density of 1. 300 g cm−3 in caesium chloride.
Fruits from isogenic lines homozygous for the ‘Neverripe’ (Nr) mutation do not ripen normally and contain reduced amounts of polygalacturonase. Only polygalacturonase 1 is produced in Nr fruit. Tomatoes from isogenic lines homozygous for the ‘ripening inhibitor’ (rin) mutation do not ripen normally and produce very little detectable polygalacturonase.
Although polygalacturonases 1 and 2 have different properties they both give rise to a single polypeptide on electrophoresis in polyacrylamide gels in the presence of sodium dodecylsulphate (Mr= 46000). A comparison of the major fragments produced by limited proteolysis of polygalacturonase 1 and 2 with chymotrypsin suggests that the polypeptides from the two isoenzymes are similar. The same conclusion was reached from a comparison of polygalacturonase 1 and 2 by radioimmunoassay, using antibody prepared against polygalacturonase 2 and 125I‐labelled polygalacturonase 2.
The results from radioimmunoassay of extracts from green and ripening fruits suggest that the increase in polygalacturonase activity during ripening is due to net synthesis of protein.
Cathepsin‐D‐Dependent Initiation of the Hydrolysis by Lysosomal Enzymes of Apoprotein B from Low‐Density Lipoproteins
Authors: VAN DER WESTHUYZEN Deneys R. , GEVERS Wieland , COETZEE Gerhard A. 
Published: March 2005

The degradation of 135I‐apoprotein B of human low‐density lipoprotein by cell extracts of cultured bovine aortic smooth muscle cells was determined by measuring the formation of acidsoluble products and by analyzing the electrophoretic patterns of digested apoprotein in gels containing sodium dodecyl sulfate. Degradation resulted in an initial rapid accumulation of a limited number of distinct smaller fragments. Two products with apparent molecular weights of 220000 and 200000 predominated. Pepstatin inhibited proteolysis almost completely, as measured by either assay.Leupeptin decreased hydrolysis to acid‐soluble products by approximately 50%, but had no effect on the initial cleavage of intact apoprotein B. Similar results were found in the case of extracts from cultured human skin fibroblasts and from adult bovine arterial smooth muscle. Leupeptin inhibited intracellular degradation of 125I‐apoprotein B in cultured cells by approximately 50%. It is concluded that the intralysosomal degradation of apoprotein B involves an initial limited endoproteolytic attack at susceptible sites by cathepsin D. This and other enzymes, including cathepsin B, then act synergistically to bring degradation to completion.
Expression of Bacteriophage M13 DNA in vivo
Authors: SMITS Mari A. , SCHOENMAKERS John G. G. , KONINGS Ruud N. H. 
Published: March 2005

Hybridization studies have indicated that on the M13 genome there are located regions which are transcribed in vivo with a high as well as with a low frequency. The region with the highest transcriptional activity coincides with the region coding for the polypeptides which during the infection cycle are needed most abundantly (gene V and gene VIII protein). The hybridization studies furthermore have indicated that at least a number of the promoters previously detected in vitro are operative in the infected cell. In addition the experimental data are consistent with the presence of three transcription termination signals on the M13 genome.With the aid of analytical and preparative hybridization studies we have been able to establish that during the infection cycle at least eleven M13‐specific RNA species are made which range in size from 280 to 2000 nucleotides. The six major RNA species (i.e. an 8‐S RNA with 370 nucleotides, a 9‐S with 420, three 11‐S species with 760, 810 and 860 nucleotides and a 14‐S species with 1140 nucleotides) are encoded by the region with the highest transcriptional activity (located between the N‐terminal end of gene X and the C‐terminal end of gene VIII). Characterization of these RNA species with respect to their genetic origin, codogenic properties and nucleotide sequences has indicated that their synthesis is terminated at the previously detected rho‐independent transcription termination signal T0.25, which is located immediately after gene VIII. From the data obtained it furthermore could be deduced that the 5′ termini of these RNA species are located as follows: for the 8‐S RNA immediately in front of gene IX (promoter G0.18), the 9‐S RNA within or immediately distal to the 5′ end of gene VII, the three 11‐S RNAs within the C‐terminal end of gene X and the 14‐S RNA immediately in front of gene X. Analyses of the 5′ ends of the major RNA chains synthesized in vivo have indicated that four (i.e. the 9‐S and three 11‐S species) out of the six RNA species are the result of processing of precursor molecules. For this processing the RNA processing enzymes RNase III, RNase E, and RNase P are not required. The meaning of these results, in terms of the mechanisms by which the expression of the M13 genome is regulated, is discussed.
Specific Protein Phosphorylation during Stimulation of Amylase Secretion by β‐Agonists or Dibutyryl Adenosine 3′,5′‐Monophosphate in the Rat Parotid Gland
Authors: JAHN Reinhard , UNGER Clemens , SÖLING Hans‐Dieter 
Published: March 2005

The present study was undertaken in order to examine the possible involvement of protein phosphorylation during β‐adrenergic stimulation in the rat parotid gland. Isolated parotid gland slices were stimulated by either isoproterenol or dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) in the presence or absence of propranolol. Amylase output was measured as a parameter for the degree of stimulation of secretion.Stimulation of secretion by either isoproterenol or Bt2AMP was associated with phosphorylation of three protein bands as revealed by sodium dodecylsulfate/polyacryla:nide gel electrophoresis and autoradiography. The apparent molecular weights of the three proteins were 35100 (protein I), 25 700 (protein II) and 20400 (protein II).After cell fractionation by differential and gradient centrifugation, protein I was enriched in a light membrane fraction most likely corresponding to the plasma membrane as revealed by marker enzyme analysis. Proteins II and III were recovered in a denser fraction containing mainly mitochondria and rough micrcsomes. The effect of isoproterenol but not that of Bt2cAMP on phosphorylation of all three protein bands was completely abolished by propranolol.The different time course in the stimulation of amylase secretion by isoproterenol and Bt2cAMP respectively was reflected by corresponding differences in the time course of protein phosphorylation.
Inducible β‐Xyloside Permease as a Constituent of the Xylan‐Degrading Enzyme System of the Yeast Cryptococcus albidus
Authors: KRÁTKÝ Zdeněk , BIELY Peter 
Published: March 2005

The yeast, Cryptococcus albidus, depending on whether it is grown on xylan or glucose, differs remarkably in the ability to take up inducers of extracellular endo‐1,4‐β‐xylanase synthesis. In washed, glucose‐grown cells the initially low ability to take up xylobiose or methyl β‐d‐xylopyranoside, increases during incubation with these compounds after a lag‐phase shorter than the induction time of the extracellular β‐xylanase. Using of methyl β‐d‐[U‐14C]xylopyranoside as a very slowly metabolizable inducer of β‐xylanase it has been established that the increase of the rate of xylobiose or methyl xyloside uptake is due to induction of an active transport system for methyl β‐d‐xyloside and β‐1,4‐xylooligosaccharides. The system is called β‐xyloside permease. The permease activity of induced cells decreases in the absence of β‐xylanase inducers. The induction of permease as well as its inactivation (degradation) can be prevented with cycloheximide, thus both events appear to be dependent on de novo protein synthesis. In analogy with other active transport systems, β‐xyloside permease function can be effectively blocked by inhibitors of energy metabolism in the cells.The demonstrated example of induction of a permease, for inducers and products of hydrolysis of an extracellular polysaccharide hydrolase, points to a new feature of induction of extracellular enzymes in eucaryotic microorganisms.
Modification of Carboxylate Groups in Bovine Pancreatic Phospholipase A2
Authors: FLEER Eduard A. M. , VERHEIJ Hubertus M. , de HAAS Gerard H. 
Published: March 2005

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water‐soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp‐39 and Asp‐99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp‐39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost.Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp‐49, demonstrating the essential role of Asp‐49 in Ca2+ binding.Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp‐49 has an apparent pK of 5.2.
The Electrochemical Proton Gradient Generated by the Fumarate‐Reductase System inEscherichia coli and Its Bioenergetic Implications
Authors: HELLINGWERF Klaas J. , BOLSCHER Jan G. M. , KONINGS Wil N. 
Published: March 2005

Proton translocation, coupled to electron transfer in the fumarate reductase system, generates and electrochemical potential gradient for protons (). The magnitude of this free energy gradient has been determined in theEscherichia coli strains ML 208–225 and AN 283. The measurements were performed in (inverted) membrane particles, right‐side out membrane vesicles and EDTA‐treated intact cells in external media of various ionic compositions and pH.The maximal values of in these three systems were +103, −101 and −105 mV, respectively. This implicates that in E. coli, upon transition from oxygen to fumarate as electron acceptor, the magnitude of the decreases considerably. This change of has substantial consequences for the cellular metabolism.
Frequency and Diversity of RNA Sequences in Different Cell Types of the Fungus Schizophyllum commune
Authors: ZANTINGE Bert , HOGE J. Harry C. , WESSELS Joseph G. H. 
Published: March 2005

In the basidiomycete Schizophyllum commune an RNA complexity of 13500 average‐sized (1100 nucleotides) RNA sequences was found by means of RNA excess hybridization to complementary DNA. 600 abundant poly(A)‐containing RNA sequences were present in about 100 copies per cell, whereas the rare 13000 poly(A)‐containing RNA sequences were present in about 5 copies per cell on average.Homologous and heterologous cDNA hybridizations to poly(A)‐rich RNAs revealed some quantitative, but no significant qualitative differences between the monokaryotic and the dikaryotic cell type. Heterologous hybridization and subsequent hybridizations of remaining single‐stranded cDNA to poly(A)‐rich RNAs from both cell types showed qualitative differences amounting to 2–3% of the rare sequences in the two cell types. However, from the results of similar hybridizations with total RNAs, i.e. including non‐polyadenylated RNA, it could be calculated that the RNA sequences in monokaryons and dikaryons are at least 99% the same.Identity of RNA sequences in monokaryons and dikaryons was also inferred by comparison of the patterns of polypeptides synthesized by translation of total RNA in cell‐free systems.
Identification in Xenopus laevis of a Class of Oocyte‐Specific Proteins Bound to Messenger RNA
Authors: DARNBROUGH Christopher H. , FORD Peter J. 
Published: March 2005

Proteins associated with poly(A)‐rich RNA in Xenopus laevis oocytes have been identified in immature ovary homogenates using sucrose gradient centrifugation, oligo(dT)‐cellulose fractionation and analysis by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and two‐dimensional acid/urea/sodium dodecyl sulphate gel electrophoresis. At least eight proteins are associated with ribonucleoprotein complexes and have the following properties.

Four major proteins, mRNP1–4, cosediment with poly(A)‐rich RNA at 40–200 S, bind to oligo(dT)‐cellulose and are oocyte‐specific. These are very basic proteins with molecular weights of about 50 × 103, 52 × 103, 56 × 103 59 × 103.
Two further proteins, mRNP5,6, molecular weights 75 × 103 and about 100 × 103, also co‐isolate with poly(A)‐rich RNA. mRNP7(Mr, 16 × 103) cosediments with poly(A)‐rich RNA but fails to bind to oligo(dT)‐cellulose. mRNP8 (Mr, 22 × 103) binds to oligo(dT)‐cellulose but sediments more slowly than the bulk of poly(A)‐rich ribunucleoprotein, at 30–70 S.
mRNP1–4 are present in immature oocytes but only mRNP3–4 are found in full‐grown oocytes, while none of them could be detected in Xenopus liver or reticulocytes. These proteins are largely cytoplasmic, associated with free mRNP particles, and differ in molecular weight from proteins isolated from polysomal mRNPs in somatic tissues.
mRNP1–4 are very abundant in immature oocytes; sedimentation properties are consistent with a protein: RNA ratio of 4:1 (w/w). The sedimentation constant of mRNP particles is resistant to concentrations of ribonuclease A which degrade ribosomes, although mRNA isolated from these ribonuclease‐treated particles appears to be partially degraded.

The role of ribonucleoprotein complexes in the stability and storage of mRNA during oogenesis is discussed.
Purification and Some Properties of NAD‐Glycohydrolase from Conidia of Neurospora crassa
Authors: MENEGUS Faustino , PACE Mario 
Published: March 2005

NAD‐glycohydrolase from conidia of Neurospora crassa was purified by affinity chromatography, using 4‐methylnicotinamide adenine dinucleotide as ligand immobilized onto Sepharose through a hydrophilic spacer arm.The pure enzyme is a glycoprotein with an isoelectric point of 5.5 and a molecular weight of 33000 as determined by sodium dodecylsulphate gel electrophoresis.The specific activity is the highest so far found for NAD‐glycohydrolases and in various aspects the enzyme is different from that isolated from mycelia ofN. crussa grown in a ‘zinc‐deficient’ medium.
Substrate‐Binding Isotherms of Spinach Chloroplastic Fructose‐1,6‐bisphosphatase and the Photoregulation of the Calvin Cycle
Authors: MEUNIER Jean‐Claude , BUC Jean , SOULIÉ Jean‐Michel , PRADEL Jacques , RICARD Jacques 
Published: March 2005

Fluorescence titration experiments of chloroplastic, fructose‐1,6‐bisphosphatase by fructose bisphosphate and magnesium have been effected using the inactive dimeric, the inactive tetrameric and the active tetrameric enzyme forms.Magnesium binding to the inactive dimeric enzyme exhibits a positive cooperativity whereas fructose 1,6‐bisphosphate exhibits no cooperativity at all. The binding of either magnesium or fructose bisphosphate to the inactive oxidized tetramer exhibits a succession of negative and positive cooperativities (mixed cooperativity). Upon reduction of the inactive tetramer by dithiothreitol and activation, the ligand binding properties of the enzyme are changed. Magnesium and fructose bisphosphate are bound to the active enzyme with a positive cooperativity.Non‐linear least‐square fitting allows one to estimate the binding constants, and therefore the free energy of binding of either magnesium or fructose bisphosphate to the various forms of the enzyme. Whatever the state of fructose‐ 1,6‐bisphosphatase, one ligand is bound per subunit. The affinity constants of magnesium for the active or inactive enzyme forms are much greater than those of fructose bisphosphate. This suggests that the metal ion gives to the enzyme the right conformation to bind the sugar phosphate.
Regulation of the Synthesis of Ribulose‐1,5‐Bisphosphate Carboxylase and Its Subunits in the Flagellate Chlorogonium elongatum
Authors: BOEGE Frank , WESTHOFF Peter , ZIMMERMANN Kurt , ZETSCHE Klaus 
Published: March 2005

Shifting of heterotrophically cultured cells of Chlorogonium elongatum to autotrophic culture conditions results in a more than tenfold increase in the level of the chloroplastic enzyme ribulose‐1,5‐bisphosphate carboxylase. This increase in enzyme amount is caused by an increased synthesis of the enzyme de novo under autotrophic culture conditions, whereas the rate of enzyme degradation is low and very similar under both growth conditions.The synthesis of the enzyme is mainly under the control of two factors. Light promotes the synthesis, while acetate, the carbon source of this organisms, has a strong inhibitory effect on this process.Since a mutant lacking chlorophyll shows a normal response to the change of culture conditions the chlorophylls can be ruled out as photoreceptors.