bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of diamines on ornithine decarboxylase activity in control and virally transformed mouse fibroblasts
Authors: Bethell D R , Pegg A E
Published: August 2015

1. The induction of ornithine decarboxylase activity in mouse 3T3 fibroblasts or an SV-40 transformed 3T3 cell line by serum was prevented by addition of the naturally occurring polyamines putrescine (butane-1,4-diamine) and spermidine. Much higher concentrations of these amines were required to fully suppress ornithine decarboxylase activity in the transformed SV-3T3 cells than in the 3T3 fibroblasts. 2. Synthetic alpha omega-diamines with 3–12 carbon atoms also prevented the increase in ornithine decarboxylase activity induced by serum in these cells. The longer chain diamines were somewhat more potent than propane-1,3-diamine in this effect, but the synthetic diamines were less active than putrescine in the 3T3 cells. There was little difference between the responses of 3T3 and SV-3T3 cells to the synthetic diamines propane-1,3-diamine and heptane-1,7-diamine. 3. These results are discussed in relation to the control of polyamine synthesis in mammalian cells.
Heterogeneity and properties of hepatic dexamethasone-binding proteins
Authors: Liu S L H , Webb T E
Published: August 2015

Evidence from experiments in vivo and in vitro is presented for the presence of three species of dexamethasone-binding proteins in rat liver, which are identified by chromatography on Sepharose 6B or by isoelectric focusing. Although two of these species (DI and DII) possess properties characteristic of a true receptor, the third binding protein (i.e. DIII), which migrates most slowly on Sepharose 6B, but has stability properties similar to protein DII, exhibits a 3-fold lower affinity for dexamethasone and the activated complex neither binds to DNA-cellulose nor translocates to the nucleus. Only the predominant liver receptor (DI), which is eluted first from Sepharose 6B, is present in Novikoff-hepatoma cytosol, suggesting that the major and minor species are not interconverted through simple dissociation during their isolation. The binding activities of all three species in the liver cytosol increase approx. 2-fold in vivo after adrenalectomy and show a transient 2-fold fall in vivo after the administration of cortisol. These changes in vivo in protein DIII shows a marked lag compared with those in proteins DI and DII, which change in parallel. It is therefore proposed that rat liver cytosol contains two dexamethasone receptors and a dexamethasone-binding protein that may be derived from these receptors.
Studies on the inhibition of protein synthesis by selenodiglutathione
Authors: Vernie L N , Collard J G , Eker A P , de Wildt A , Wilders I T
Published: August 2015

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.
Re-examination of the subcellular localization of thyroxine 5′-deiodination in rat liver
Authors: Auf Dem Brinke D , Hesch R D , Köhrle J
Published: August 2015

We describe the existence of at least two thyroxine 5′-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5′-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5′-deiodination. However, this process is not mediated by only a single enzyme.
Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A
Authors: Tipping E , Ketterer B , Christodoulides L
Published: August 2015

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we examined the interactions of bromosulphophthalein, oestrone sulphate, haem and bilirubin with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylchone/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. In all four cases, saturation effects were observed. Values of Vmax (v = mol of compound bound/mol of lipid phosphorus) at 25 degrees C were: for bromosulphophthalein, approximately 0.1; for oestrone sulphate, approximately 0.25; for haem, approximately 0.25 (all at pH 7.4); and for bilirubin 0.1–0.2 (at pH 8.2). 3. Limiting values of v/c (c = unbound concentration) as v leads to 0 at 25 degrees C and pH 7.4 are: for bromosulphophthalein, 6.25 × 10(4) litre-mol-1; for oestrone sulphate, 7.8 × 10(2) litre-mol-1; for haem, 4.5 × 10(5) litre-mol-1; and for bilirubin, approximately 1.2 × 10(4) litre-mol-1. For haem the result depends on the assumption that only the monomeric form binds to the lipid. 4. The binding of each compound was decreased by cholesterol; bromosulphophthalein and oestrone sulphate were affected more than haem and bilirubin. 5. Bromosulphophthalein at saturating concentration decreased the limiting values of v/c of the other three compounds by approximately one order of magnitude. 6. By assuming that the interactions with egg phosphatidylcholine resemble those with the phospholipid components of mammalian intracellular membranes the binding data for phosphyatidylcholine, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 92, 51, 98 and 47% of the total bromosulphophthalein, oestrone sulphate, haem and bilirubin respectively may be membrane-bound.
Lower rates of protein degradation in developing rat brain
Authors: Dahl J L , Weibel V J
Published: August 2015

Protein-degradation rates in developing rat brain were estimated from the decay in total radioactivity in proteins labelled by a single intraperitoneal injection of NaH14CO3 to 5-day-old animals. In contrast with previous reports, our results indicate that degradation rates are lower in developing than in adult brain and suggest that in brain, as has been observed in liver, adrenal gland, muscle, cultured mammalian cells and bacteria, reduced rates of protein degradation contribute to the increase in protein content under conditions of rapid growth.
Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate
Authors: Smith M J , Schreiber J B , Wolf G
Published: August 2015

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.
Ability of human chorionic gonadotropin β-subunit to inhibit the steroidogenic response to lutropin
Authors: Dighe R R , Muralidhar K , Moudgal N R
Published: August 2015

Ability of the beta-subunit of human chorionic gonadotropin to inhibit the response to lutropin (luteinizing hormone, LH) was tested in the immature rat ovarian system and pregnant-mare-serum-gonadotropin-primed rat ovarian system with progesterone production being used as the response. Human chorionic gonadotropin beta-subunit was found to inhibit human and ovine lutropin-stimulated progesterone production. At a constant dose of lutropin, inhibition was dependent on the concentration of beta-subunit. When concentration of the beta-subunit was kept constant at 5.0 microgram/ml and the concentration of lutropin was varied, the inhibition was maximum at the saturating concentration of the native hormone. The alpha-subunit of the human chorionic gonadotropin did not inhibit the response to lutropin. The lutropin/beta-subunit ratio required to produce an inhibition of response was much lower than that required to bring about an observable inhibition of binding.
Alterations in the rate of lipogenesis in vivo in maternal liver and adipose tissue on premature weaning of lactating rats: a possible regulatory role of prolactin
Authors: Agius L , Robinson A M , Girard J R , Williamson D H
Published: August 2015

Removal of pups for 24 h from rats at peak lactation decreased 3H2O incorporation into lipid in vivo in mammary gland by 95%, whereas it was increased in liver (77%) and adipose tissue (330%). These increases were prevented by administration of prolactin. Plasma insulin increased 3-fold on weaning and this was partially prevented by prolactin.
Assay of adenosine 3′-phosphate 5′-sulphatophosphate in hepatic tissues
Authors: Wong K P , Yeo T
Published: August 2015

A fluorimetric assay, based on the ability of boiled hepatic extracts to support the sulphO-conjugation of harmol, was used to demonstrate and quantify PAdoPS (adenosine 3′-phosphate 5′-sulphatophosphate) present in liver. A stoicheiometric relationship was established between the sulphate conjugate formed and the ‘active sulphate’ utilized. Guinea-pig, rat, mouse and rabbit livers contain 3.3, 2.9, 0.8 and 0.5 mumol of PAdoPS/100 G wet wt. respectively.
Preparation and properties of amphipathic enzyme-polymer conjugates
Authors: Smith R A G
Published: August 2015

Amphipathic enzyme-polymer conjugates were prepared by reaction of hydrophilic enzymes with an anhydride polymer partially substituted with pendant hydrophobic groups. The products formed non-covalent aggregates of high molecular weight, dissociable by non-ionic detergents and urea and subject to additional aggregation at high ionic strength. Oil-in-water emulsions formed in the presence of such conjugates were shown to possess enzyme activity associated with the surface of the water-immiscible liquid. Fluorochrome labelling of conjugates showed that binding occurred at the surface of solvent droplets, and the conjugates were also found to aggregate liposomes.
Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of γ-glutamyl kinase
Authors: Krishna R V , Leisinger T
Published: August 2015

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.
Inhibition of human alkaline phosphatases by vanadate
Authors: Seargeant L E , Stinson R A
Published: August 2015

Orthovanadate was shown to be a potent competitive inhibitor (Ki less than 1 microM) of purified alkaline phosphatase from human liver, intestine of kidney. Inhibition was reversed and full enzymic activity restored in the presence of 1mM-adrenaline. Phosphate and vanadate competed for the same binding site on the enzyme.
The development of SS′-polymethylenebis(methanethiosulphonates) as reversible cross-linking reagents for thiol groups and their use to form stable catalytically active cross-linked dimers within glyceraldehyde 3-phosphate dehydrogenase
Authors: Bloxham D P , Sharma R P
Published: August 2015

The synthesis of a series of SS′-polymethylenebis(methanethiosulphonates) including the pentane, hexane, octane, decane and dodecane derivatives is described. These derivatives were synthesized by condensation between dibromoalkanes and potassium methanethiosulphonate in refluxing methanol and this seems an especially versatile reaction for the synthesis of asymmetric thiosulphonate derivatives. The synthesis of SS′-[1,8-3H4]-octamethylenebis(methanethiosulphonate) was also perfomed. Cross-linking was demonstrated in the four enzymes lactate dehydrogenase, phosphofructokinase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase. For all four enzymes cross-linking was efficiently reversed by reducing conditions in denaturing solvents. The reaction with glyceraldehyde 3-phosphate dehydrogenase was unique in that only the cross-linked dimer was produced in significant amounts (greater than 90% of total products as dimer). This reaction was followed in detail with radioactive cross-linking reagent. Inhibition of enzyme activity was extremely fast and showed an asymmetric distribution of enzyme activity on subunits. Thus complete modification of only one subunit resulted in up to 75% inhibition of enzyme activity. Reaction of glyceraldehyde 3-phosphate dehydrogenase with 1.25 mol of SS′-octamethylenebis(methanethiosulphonate) per mol of enzyme subunit produced two species of protein. The first species was obtained in 20% yield and was only partially re-activated on mild reduction with 2-mercaptoethanol. The second species was isolated in 66% yield and was completely re-activated on mild reduction. Before reduction there was 4 mol of inhibitor per tetramer for the latter species, and more than 95% of the enzyme was present as a dimer on non-reducing electrophoresis. After mild reduction 2 mol of inhibitor was still bound per tetramer, the enzyme was now catalytically active and the dimer was still the major structure on non-reducing electrophoresis. Thus mild reduction of SS′-octamethylenebis(methanethiosulphonate-treated glyceraldehyde 3-phosphate dehydrogenase enabled the production of active enzyme in which there is a stable cross-link across one of the molecular axes of the tetrameric enzyme. This cross-link was only reversed if reduction was performed when the enzyme was denatured. The molecular weight of cross-linked and re-activated cross-linked glyceraldehyde 3-phosphate dehydrogenase was established as 144000 (tetramer) by sucrose-density-gradient centrifugation. These observations are interpreted in terms of the molecular structure of glyceraldehyde 3-phosphate dehydrogenase.
Isolation and partial characterization of three acidic proteinases in erythrocyte membranes
Authors: Pontremoli Sandro , Salamino Franca , Sparatore Bianca , Melloni Edon , Morelli Alessandro , Benatti Umberto , De Flora Antonio
Published: August 2015

1. The distribution of proteolytic activity in membranes from human erythrocytes and from rabbit reticulocytes and erythrocytes was investigated, after removal of leucocytes and platelets from the cell suspensions. 2. All membrane preparations displayed proteolytic activity in the acidic pH region only. Membranes from human and rabbit mature erythrocytes showed latent activity, which could be increased when extracted with a number of detergents. 3. Three active fractions were resolved either by gel chromatography of solubilized membrane extracts or by standard polyacrylamide-gel electrophoresis. The three proteinase activities (designated proteinases I, II and III) were purified from solubilized extracts of human erythrocyte membranes. 4. The relevant mol.wts. were around 80000, 40000 and 30000, respectively, and each of the three proteinases appeared to be composed of a single polypeptide chain. 5. Distinctive pH optima (in the range pH2.8–3.9) and different saturation profiles with globin as substrate were observed for proteinases I, II and III. 6. Dithioerythritol, Hg2+ and Cu2+ inhibited each of the three human enzymes, but more selective inhibitory effects were exerted by other modifiers of proteolytic enzymes and by haemin. Similar effects were observed with the three proteinases from rabbit cells. 7. The activity of the three human proteinases seems to be restricted to naturally occurring protein substrates, although with poor specificity, and none of them was active on synthetic substrates. 8. Digestion of globin by each of the three enzymes yielded similar polypeptide fragments in all cases, this indicating an endopeptidase type of activity.
Properties of allophycocyanin II and its α- and β-subunits from the thermophilic blue–green alga Mastigocladus laminosus
Authors: Gysi Jürg R. , Zuber Herbert
Published: August 2015

Purified allophycocyanin II and its subunits have been examined with respect to spectroscopic properties, sedimentation, reconstitution and isoelectric behaviour. In 0.02m-potassium phosphate buffer, pH8.0, and at a concentration of 0.25mg/ml, allophycocyanin II and its α- and β-subunits show visible absorption maxima at 650, 615 and 615nm respectively, whereas the fluorescence emission maxima were determined to be at 662, 640 and 630nm respectively. The absorption difference spectrum (dilution difference) of allophycocyanin II displays maxima at 650 and 590nm with a minimum at 610nm. The c.d. spectrum of allophycocyanin II showed only one positive-ellipticity band at 635nm, and a major negative-ellipticity band at 340nm. Oxidation of allophycocyanin II, low- and high-pH solutions (pH3.0 and 11.0), various ethanol concentrations as well as dialysis against distilled water induce a spectral change leading to phycocyanin-like characteristics. In most cases these shifts are reversible. Allophycocyanin II is thermostable over a period of 60min at temperatures up to 60°C. The isoelectric points of allophycocyanin II and its α- and β-subunits are 4.65, 4.64 and 4.82 respectively. Estimated molecular weights from sedimentation-equilibrium analyses were 102500 for allophycocycanin II, 16000 for the α- and 31500 for the β-subunit. Recombination of α- and β-subunits leads to allophycocyanin II, which is indistinguishable from native allophycocyanin with respect to its spectral form, to its gel-filtration and to its electrophoretic behaviour.
Bi- and tri-antennary human transferrin glycopeptides and their affinities for the hepatic lectin specific for asialo-glycoproteins
Authors: Hatton Mark W. C. , März Leopold , Berry Leslie R. , Debanne Maria T. , Regoeczi Erwin
Published: August 2015

Glycopeptides were isolated from a proteolytic digest of human transferrin. After mild acid hydrolysis the desialylated glycopeptides were labelled by the galactose oxidase/NaB3H4 procedure and then fractionated by Sephadex-gel filtration or by anion-exchange chromatography. Either technique allowed separation of the two heterosaccharide chains (designated glycan I and glycan II) previously described for this protein by Spik, Vandersyppe, Fournet, Bayard, Charet, Bouquelet, Strecker & Montreuil (1974) (in Actes du Colloque Internationale No. 221 vol. 1, pp. 483–499). Subsequent chromatography on Sepharose–concanavalin A separated fractions containing different quantities of carbohydrates for each glycan, as indicated by analyses. The isolated glycan fractions were then tested for their abilities to bind to the immobilized rabbit hepatic lectin. Our studies suggest that either glycan can have a bi- or tri-antennary structure. Desialylated biantennary glycans I and II did not bind to the hepatic lectin. Desialylated triantennary glycan I was slightly retarded by the hepatic lectin, whereas the triantennary glycan II consisted of equal quantities of a retarded and a bound type. Desialylated triantennary glycan II was totally displaced from the hepatic lectin by using a buffer containing 0.05m-EDTA. The results suggest that greater structural heterogeneity exists in the carbohydrate moiety of human transferrin than was previously envisaged. Such heterogeneity could be reflected in several molecular forms of human transferrin, which, after desialylation, differ significantly in their affinities for the hepatic lectin.
The isolation and partial sequence of peptides produced by cyanogen bromide cleavage of calf thymus non-histone chromosomal high-mobility-group protein 2. Sequence homology with non-histone chromosomal high-mobility-group protein 1
Authors: Walker J M , Gooderham K , Johns E W
Published: August 2015

Peptides produced by CNBr cleavage of non-histone chromosomal protein HMG 2 (CNBr peptides) were isolated and characterized, and their partial sequences were determined. The present sequence data account for over half of the sequence of the protein HMG (high-mobility-group) 2 molecule, and, together with previously published results, provide interesting information on the charge distribution within the molecule. Comparison of the CNBr-peptide-sequence data for protein HMG 2 with the previously published data on the CNBr peptides from protein HMG 1 reveals extensive sequence homology between the two proteins. Detailed evidence for the amino acid-sequence data has been deposited as Supplementary Publication SUP 50095 (6 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.
An interaction between lysozyme and mucus glycoproteins. Implications for density-gradient separations
Authors: Creeth J M , Bridge J L , Horton J R
Published: August 2015

1. Some mucus glycoproteins form soluble complexes with lysozyme at neutral pH values. 2. The extent of complex-formation was determined, by an ultracentrifugal difference method, for a range of glycoproteins covering the common blood-group specificities. 3. Interaction was strongest with those glycoproteins of blood-group Lea specificity; these were also richest in sialic acid. 4. Interaction diminished with increase of ionic strength, and was not detectable at I 0.50; however, an asialoglycoprotein was found to retain some activity. The interaction is accordingly primarily, but probably not exclusively, coulombic in origin. 5. The buoyant density of lysozyme in CsCl, CsBr, CsI and Cs2SO4 was determined; the values in the last three salts are anomalously high. This finding accounts for the previously noted difficulty of separating free protein from glycoproteins by single-stage centrifugation in CsBr. 6. Conditions for effective separation of glycoproteins from secretions containing lysozyme by density-gradient centrifugation are reported.
Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro
Authors: Grankvist K , Marklund S , Sehlin J , Täljedal I B
Published: August 2015

Experiments with isolated pancreatic islets or dispersed islet cells from non-inbred ob/ob mice were performed to test the hypothesis that free radicals, notably OH., mediate the diabetogenic toxicity of alloxan. Accumulation of 86Rb+ by whole islets and exclusion of Trypan Blue by dispersed cells were used as previously validated criteria of islet-cell viability. Alloxan alone drastically inhibited the Rb+ accumulation and significantly decreased the frequency of cells excluding Trypan Blue. Enzymic scavengers of O2.- and H2O2 or non-enzymic scavengers of OH. or singlet oxygen were added to the incubation medium and tested for their ability to protect against these effects of alloxan. Superoxide dismutase, catalase, dimethyl sulphoxide, benzoate, and mannitol counteracted the effects of alloxan in both cytotoxicity assays. Significant protection of the Rb+-accumulating capacity was also afforded by butanol, caffeine, theophylline, NADH, NADPH and, to a small extent, NAD+. Urea has a poor affinity for OH. and did not protect against alloxan. No effect was obtained with the singlet-oxygen scavenger, histidine. Except for the protection by NADH and NADPH, which may be due to a direct reaction with alloxan in the medium, the results strongly support the hypothesis. beta-Cells may be particularly vulnerable to alloxan because their metabolic specialization facilitates reduction of the drug and perhaps of other substrates for O2.–yielding redox cycles.
Clofibrate-induced increase in coenzyme A concentration in rat tissues
Authors: Voltti Hilkka , Savolainen Markku J. , Jauhonen V. Pekka , Hassinen Ilmo E.
Published: August 2015

1. When clofibrate [ethyl 2-(4-chlorophenoxy)-2-methylpropionate] was administered subcutaneously to rats (600mg/kg per day for 5 days), the concentration of CoA and its acyl derivatives increased in several tissues. The increase in total CoA was 3.2-fold in the liver, 1.8-fold in the kidney, 2.7-fold in the heart and 2.4-fold in skeletal muscle. 2. To study the mechanism of this phenomenon, clofibrate-treated rats were injected with [3H]pantothenate intracardially and killed after 15min, 30min, 1 and 2h and 1, 3, 5 and 7 days for the determination of the incorporation of radioactivity into CoA and its precursors. Incorporation into CoA after 2h was 6.2-fold in the liver as compared with the control values and 4.6-fold in the kidneys. 3. The disappearance of the label from CoA was very slow compared with the rate of incorporation; it exhibited exponential kinetics, and was slower in the livers of the clofibrate-treated rats (t½ 18.2 days) than in the controls (t½ 5.6 days). 4. The rate of CoA degradation, calculated from the calculated rate constants of the apparent first-order kinetics of the disappearance of the label and from the CoA pool sizes, was approximately the same in the clofibrate-treated animals (11.5pmol/min per g), and the controls (11.6pmol/min per g). 5. These rates of CoA degradation indicate that the effect of clofibrate on CoA concentration may be mainly due to inhibition of the enzymes of CoA degradation, although recycling of the label cannot be excluded. The increase in the rate of pantothenate incorporation into CoA suggests that clofibrate also increases the synthesis of CoA.
Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo
Authors: Quaroni Andrea , Kirsch Katharina , Weiser Milton M.
Published: August 2015

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-3H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.
Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex
Authors: Lücke Heinrich , Stange Gertraud , Murer Heini
Published: August 2015

Uptake of SO42− into brush-border membrane vesicles isolated from rat kindey cortex by a Ca2+-precipitation method was investigated by using a rapid-filtration technique. Uptake of SO42− by the vesicles was osmotically sensitive and represented transport into an intra-vesicular space. Transport of SO42− by brush-border membranes was stimulated in the presence of Na+, compared with the presence of K+ or other univalent cations. A typical ‘overshoot’ phenomenon was observed in the presence of an NaCl gradient (100mm-Na+ outside/zero mm-Na+ inside). Radioactive-SO42− exchange was faster in the presence of Na+ than in the presence of K+. Addition of gramicidin-D, an ionophore for univalent cations, decreased the Na+-gradient-driven SO42− uptake. SO42− uptake was only saturable in the presence of Na+. Counter-transport of Na+-dependent SO42− transport was shown with MoO42− and S2O32−, but not with PO42−. Changing the electrical potential difference across the vesicle membrane by establishing different diffusion potentials (anion replacement; K+ gradient±valinomycin) was not able to alter Na+-dependent SO42− uptake. The experiments indicate the presence of an electroneutral Na+/SO42−-co-transport system in brush-border membrane vesicles isolated from rat kidney cortex.
Studies on sex-organ development. Changes in the oestrogenic response of the chick Müllerian duct as measured by chromatin template and ribonucleic acid initiation capacity
Authors: Andrews G K , Teng C S
Published: August 2015

Assays of transcription in vitro, with Escherichia coli RNA polymerase or wheat-germ RNA polymerase II, were used to characterize chromatin templates isolated from the left Müllerian duct of the chick embryo during normal development, and during development in the presence of diethylstilboestrol. Control Müllerian-duct template capacity with E. coli RNA polymerase decreased from 6.42% on day 10 to 4.34% by day 15 of development. Similar results were found with wheat-germ RNA polymerase II. In the presence of rifampicin and heparin, the prokaryotic enzyme transcribed a number-average RNA chain of 670 nucleotide residues, at an average rate of 110 nucleotide residues/min, from Müllerian-duct chromatin of all developmental stages. From day 10 to day 15 there was a 44% decrease in the number of initiation sites for E. coli RNA polymerase on Müllerian-duct chromatin. A 47% decline was observed when these chromatins were transcribed with excess RNA polymerase II in the presence of rifamycin Af/013. Signs of increasing responsiveness to oestrogen developed between days 10 and 16. Embryos exposed to maximally responsive doses of diethylstilboestrol for 2 days showed increases in Müllerian-duct chromatin template capacity, RNA-chain initiation sites, wet weight, protein and RNA. The changes seen in the oviduct of the 1-week-old chick injected for 2 days with diethylstilboestrol were defined as 100% responses. By comparison, the Müllerian duct, after exposure to diethylstilboestrol from day 10 to day 12, from day 13 to day 15 or from day 16 to day 18, showed a 15%, 39% and 72% template response respectively, and a 42%, 56% and 85% initiation-site change respectively. A similar developmental trend was observed in all parameters. It is concluded that oestrogenic responsiveness in the developing Müllerian duct increases from day 10 to nearly maximal values by day 16 of development, and that this transition is paralleled by a progressive restriction of genomic activity.
Multiplicity of induction patterns of rat liver microsomal mono-oxygenases and other polypeptides produced by administration of various xenobiotics
Authors: Sharma R N , Cameron R G , Farber E , Griffin M J , Joly J G , Murray R K
Published: August 2015

Induction of hepatic microsomal mono-oxygenase species after administration of various xenobiotics is a well-documented phenomenon. To examine the number and specific species of rat liver microsomal membrane polypeptides involved in such responses, we have used sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to analyse microsomal fractions from animals treated with a number of important xenobiotics. The following are the principal points to have emerged from this study. 1. A minimum of twelve electrophoretically distinct patterns of induction of haemopolypeptides and other polypeptides could be distinguished after administration, either singly or in certain combinations, of phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls, 2-acetylaminofluorene, safrole (or isosafrole), pregnenolone-16 alpha-carbonitrile and ethanol. The patterns consisted of various permutations of the amounts of eight polypeptides of 47000-56000 mol.wt., of which at least three were haemopolypeptides. The possible identities of these polypeptides, which included species of cytochrome P-450, cytochrome P-448 and epoxide hydratase, are discussed. 2. Agents (3-methylcholanthrene, benzo[a]-pyrene, polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone) that result in the induction of cytochrome P-448 caused a marked increase in two polypeptides of 54000 and 56000 mol.wt., whereas safrole and isosafrole induced only the former polypeptide. 3. Administration of 2-acetylaminofluorene resulted in the induction of two polypeptides; evidence is presented that suggests that one of these is a species of epoxide hydratase [cf. Levin, Lu, Thomas, Ryan, Kizer & Griffin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3240-3243] ANd that the other may be a novel haemopolypeptide. 4. The overall results emphasize the complexity of the responses exhibited by rat liver microsomal fractions to the administration of xenobiotics.
Metabolism of glucose in hyper- and hypo-thyroid rats in vivo. Glucose-turnover values and futile-cycle activities obtained with 14C- and 3H-labelled glucose
Authors: Okajima F , Ui M
Published: August 2015

1. A trace amount of glucose labelled with 14C uniformly and with 3H at position 2, 3 or 6 was injected intravenously into starved rats to measure the turnover rate of blood glucose. 2. Reliable estimates were made based on the semilogarithmic plot of specific radioactivity of the glucose contained in whole blood samples taken from the tail vein. 3. Glucose turned over more rapidly in hyperthyroid and more slowly in hypothyroid than in euthyroid rats. The percentage contribution of glucose recycling (determined from the difference in replacement rates between [U-14C]glucose and [6-3H]glucose) to the glucose utilization increased on induction of hyperthyroidism. 4. Futile cycles between glucose and glucose 6-phosphate (determined from the difference between replacement rates of [2-3H]glucose and [6-3H]glucose) were activated and inactivated by induction of hyperthyroid and hypothyroid states respectively. 5. The hepatic content of glycogen was much lower in hyper- and hypo-thyroid than in euthyroid rats. The enhanced glucose production in hyperthyroid rats resulted from not only activationof hepatic gluconeogenesis but also diversion of the final product of gluconeogenesis from liver glycogen to blood glucose. In hypothyroidism, the inhibition of gluconeogensis led to suppression of both glucose production and glycogenesis in the liver.
The molecular compression of dextran
Authors: Ogston A G , Preston B N
Published: August 2015

The suggestion is made that, in solution, the flexible-chain molecules of dextran can undergo an osmotic compression as concentration is increased. Approaches are developed described the molecular shrinkage (i) as arising from intra- and inter-molecular forces, (ii) based on the molecular characteristics of the dextran, and (iii) as estimated by viscosity measurements. Comparison with the macroscopic shrinkage of cross-linked dextran (Sephadex) beads [Edmond, Farquhar, Dunstone & Ogston (1968) Biochem. J. 108, 755-763] is made. In all systems studied, the experimental estimates of compression, both from gel-shrinkage and viscosity measurements were in reasonable agreement with theoretical predictions. The interpretation of the viscosity concentration-dependence was applied to compact structures (albumin and Percoll). Their behaviour was in marked contrast with that of dextran. It is noted that molecular compression may be important in considering transport processes in and thermodynamic properties of concentrated systems.
Purification of a phosphoprotein from chromatin of rat liver
Authors: Chan P K , Liew C C
Published: August 2015

A simple and effective method to purify a phosphoprotein (B2) (Mr 68,000, pI 6.2-8) from phenol-soluble non-histone chromatin proteins of rat liver is described. The purification involved only two steps, CM-cellulose chromatography and preparative SDS/polyacrylamide (10%)-gel electrophoresis. The purified phosphoprotein B2 was shown to be homogeneous by SDS/polyacrylamide-gel electrophoresis. The yield was 2% of total non-histone chromatin proteins. The acidic to basic amino acid ratio of phosphoprotein B2 was less than 1, with high contents of glutamic acid, aspartic acid, arginine, lysine, glycine and alanine. The phosphate content of this protein is 0.3%.
Lectin-binding proteins in central-nervous-system myelin. Detection of glycoproteins of purified myelin on polyacrylamide gels by [3h]concanavalin A binding
Authors: McIntyre L J , Quarles R H , Brady R O
Published: August 2015

Concanavalin A strongly agglutinates purified fragments of immature and mature rat brain myelin, but only weakly agglutinates mature bovine and human myelin fragments. A sensitive method involving [3H]concanavalin binding to sodium dodecyl sulphate/polyacrylamide gels was used to detect the concanavalin A-binding proteins in purified myelin. When applied to mature rat brain myelin proteins that had been labelled in vivo with [14C]fucose, the distribution of the [3H]concanavalin A on the gel was very similar to that of [14C]fucose with the major peak corresponding to the major myelin-associated glycoprotein. The technique revealed that the immature form of the myelin-associated glycoprotein with a slightly larger apparent molecular weight also bound concanavalin A, and that in purified immature rat myelin the quantitative importance of some of the other glycoproteins in binding concanavalin A was increased relative to the myelin-associated glycoprotein. The separated proteins of bovine and human myelin bound more [3H]-concanavalin A than those of rat myelin. In these species, the myelin-associated glycoprotein was a major concanavalin A-binding protein, although two higher-molecular-weight glycoproteins also bound significant quantities of [3H]concanavalin A. The results indicate that there are receptors for concanavalin A on the surface of rat, bovine and human myelin membranes and suggest that the myelin-associated glycoprotein is one of the principal receptors.
A radiochemical titrant for the determination of the operational molarity of solutions of acid proteinases
Authors: Irvine G B , Elmore D T
Published: August 2015

N-Diazoacetyl-L-phenylalanine 3-phenyl[2,3-3H]propylamide was synthesized and shown to inhibit pepsin A (EC3,4,23.1) and cathepsin D (EC 3.4.23.5) irreversibly and stoicheiometrically in the presence of Cu2+. Quantitative separation of the inhibited enzyme from excess reagent by gel filtration followed by measurement of the radioactivity of the protein peak provided a method for determining the operational molarity of these enzymes. Several other putative active-site-directed irreversible inhibitors were synthesized, but were inactive. Data on the synthesis of these compounds have been deposited as Supplementary Publication SUP50096 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
The rate-limiting step of the protamine-induced adenosine triphosphatase activity of adenosine triphosphate-G-actin
Authors: Ferri A , Magri E , Grazi E
Published: August 2015

The release of Pi from the Pi-G-actin-ADP complex is the rate-limiting step in the ATPase activity that is shown by ATP-G-actin in the presence of protamine.
Isolation of 35S- and 3H-labelled proteoglycans from cultures of human embryonic skin fibroblasts
Authors: Cöster L , Carlstedt I , Malmström A
Published: August 2015

35SO42– and [3H]-leucine-labelled proteoglycans were isolated from the medium of a fibroblast culture, from an EDTA extract of the monolayer, and from consecutive dithiothreitol and guanidine hydrochloride extracts of the cells. Proteoglycans of different sizes were isolated from the extracts by gel chromatography on Sepharose 4B. In the medium and the EDTA extract the largest proteoglycans contained only 35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparan [35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparin [35S]sulphate. The galactosaminoglycan-containing proteoglycans of the various extracts were separated into a larger component, containing chondroitin sulphate-like side chains, and a smaller component, containing dermatan sulphate. The larger proteoglycan of the medium showed reversible association-dissociation behaviour when chromatographed on Sepharose CL2B in phosphate-buffered saline and 4M-guanidine hydrochloride respectively. This property remained after removal of extraneous proteins by CsCl-density-gradient centrifugation in guanidine hydrochloride. The association was markedly increased by the addition of high-molecular-weight hyaluronic acid.
Metabolism of palmitate in perfused rat liver. Isolation of subcellular fractions containing triacylglycerol
Authors: Kondrup J
Published: August 2015

1. The metabolism of [1-14C]palmitate in rat liver was studied in a single-pass perfusion system at concentrations of 0.2 or 1 mM. 2. After the perfusion the liver was homogenized and the floating fat was isolated. The incorporation of [1-14C]palmitate into triacylglycerol in this pool increased 9-fold when the palmitate concentration in the medium was increased from 0.2 to 1 mM. In time studies with 1 mM-[1-14C]palmitate 75% of the total accumulation of triacylglycerol occurred in this pool. Our results support the concept that the floating-fat fraction contains the storage pool of triacylglycerol, i.e. the cytoplasmic lipid droplets. 3. In a particulate preparation consisting mainly of mitochondria and microsomal fraction the incorporation of [1-14C]palmitate into triacylglycerol was proportional to the fatty acid concentration. Triacylglycerol in the perfusate medium and in the particulate fraction was in isotopic equilibrium, which indicates that the particulate fraction contained the precursor pool for secreted triacylglycerol, i.e. the pool in endoplasmic reticulum and Golgi apparatus. 4. The oxidation to labelled water-soluble products and to CO2 was increased 14-fold by the 5-fold increase in palmitate concentration.
Identification of an NAD(P)+-dependent ‘malic’ enzyme in small-intestinal-mucosal mitochondria
Authors: Sauer L A , Dauchy R T , Nagel W O
Published: August 2015

An NAD(P)+-dependent ‘malic’ enzyme is shown to be present in mitochondria from small-intestinal mucosa. The intracellular location, activity and regulatory kinetic properties of the enzyme suggest that it participates in the major energy-producing pathway for net oxidation of glutamine-derived tricarboxylic acid-cycle intermediates.
Isolation and purification of a hen nuclear oestrogen receptor and its effect on transcription of chick chromatin
Authors: Smith R G , Schwartz R J
Published: August 2015

An oestrogen receptor was isolated, characterized and purified from the nuclear fraction of the hen oviduct. The receptor sediments at 4.6 S on glycerol gradients, has an equilibrium dissociation constant (Kd) of 1.1 × 10(-10)M, an association constant (ka) of 1.4 × 10(-6) M-1.S-1, and a dissociation constant (kd) of 5 × 10(-5) s-1. The receptor chromatographed from DEAE-cellulose as a single peak at 0.15 M-KCl and was not retained by phosphocellulose. Polyacrylamide-gel electrophoresis of the receptor in the presence of sodium dodecyl sulphate demonstrated two subunits with apparent mol.wts. of 74000 and 80000. The overall purification achieved was 90000-fold by using a combination of cell fractionation, (NH4)2SO4 fractionation and affinity chromatography. This represents the first separation, isolation and purification of the highest-affinity binding component (Kd 10(-10)M) of two high-affinity oestrogen-binding proteins present in both chick and hen oviduct cytosol and nuclei. To examine directly the effect of the purified receptor on transcription a reconstituted cell-free system was used, which contained the receptor–oestradiol complex, Escherichia coli RNA polymerase, rifampicin and chromatin prepared from hormone-withdrawn chick tissue. The receptor-hormone complex at a concentration of 0.1 nM stimulated transcription of oviduct chromatin by promoting an increase of 14000 sites for RNA-chain initiation, which is similar to the number of additional sites measured in the oviducts of diethylstilboestrol-stimulated immature chicks [Tsai, Schwartz, Tsai & O'Malley (1975) J. Biol. Chem. 250, 5165-5174]. Oestradiol alone had no effect on transcription. Thus the data demonstrate that the purified nuclear oestradiol-receptor complex can regulate gene transcription in vitro in a manner similar to that observed in target cells in vivo.