bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged induction of hepatic haem oxygenase and decreases in cytochrome P-450 content by organotin compounds
Authors: Rosenberg D W , Drummond G S , Cornish H C , Kappas A
Published: August 2015

The administration of organotin compounds to rats in single doses causes a significant and prolonged induction of haem oxygenase and a sustained decrease in haemoprotein content in the liver. The extent of induction of hepatic haem oxygenase varied between 3 and 5-fold at 72h after a single injection of water-insoluble organotins of differing structure. The alterations in haem metabolism produced by tricyclohexyltin hydroxide were studied in detail. The effects were dose-dependent, with doses as low as 3.75 mg/kg body wt. resulting in significant induction of haem oxygenase and a decrease in cytochrome P-450 and cytochrome b5 contents at 72h in the liver. The effects with time of a single dose of tricyclohexyltin on various parameters of liver haem metabolism were also examined. The organotin produced a substantial and very prolonged induction of haem oxygenase accompanied by a steady decline in cytochrome P-450 content for periods up to 8 days. The long duration of action of these organotins with respect to induction of haem oxygenase and depletion of cellular haemoprotein content provides a highly sensitive metabolic system with which to define further the toxic potential of organometals as well as to study the adaptive responses in liver to long-term perturbations of haem metabolism by foreign chemicals.
The interaction between methanol dehydrogenase and the autoreducible cytochromes c of the facultative methylotroph Pseudomonas AM1
Authors: O'Keeffe D T , Anthony C
Published: August 2015

Cytochromes cH and cL were autoreduced at high pH (pK greater than 10) and the autoreduced cytochromes reacted with CO. The autoreduction was first-order with respect to oxidized cytochrome c and was reversible by lowering the pH. Pure methanol dehydrogenase reduced cytochrome c (in the absence of methanol) by lowering the pK for autoreduction to less than 8.5. A mechanism is proposed for the autoreduction of cytochrome c and its involvement in the reaction with methanol dehydrogenase.
A high-affinity cytosol binding protein for 1 α,25-dihydroxycholecalciferol in the uterus of Japanese quail
Authors: Takahashi N , Abe E , Tanabe R , Suda T
Published: August 2015

Cytosol fractions prepared from the uterine mucosa of egg-laying Japanese Quail were analysed for binding of the metabolites of cholecalciferol. When the uterus was incubated at 37 degrees C with various radioactive metabolites of cholecalciferol, the nuclear fraction incorporated only 1 alpha,25-dihydroxy[3H]cholecalciferol. When the uterus was incubated at 0 degree C with 1 alpha,25-dihydroxy[3H]cholecalciferol, most of the radioactivity was found in the cytosol. Translocation of 1 alpha,25-dihydroxy[3H]cholecalciferol from the cytosol to the nucleus was temperature-dependent. The addition of 100-fold excess amounts of unlabelled 1 alpha-25-dihydroxycholecalciferol significantly diminished the nuclear binding of 1 alpha,25-dihydroxy[3H]cholecalciferol. The cytosol fraction contained a 3.5 S macromolecule that specifically binds 1 alpha,25-dihydroxy[3H]cholecalciferol. The dissociation constant was 0.39 nM and the maximal binding was 55 fmol/mg of protein. These results strongly suggest that the uterus in egg-laying birds is a target organ or 1 alpha,25-dihydroxycholecalciferol.
Tumour calcitonin. Interaction with specific calcitonin receptors
Authors: Ham J , Ellison M L , Lumsden J
Published: August 2015

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.
Characterization of cell-surface glycopeptides from mouse cerebral cortex that inhibit cell growth and protein synthesis
Authors: Kinders R J , Milenkovic A G , Nordin P , Johnson T C
Published: August 2015

Glycopeptides were isolated from the cell surfaces of mouse brain cortical tissue that inhibited both cell division and protein synthesis by cells in culture. The protein-synthesis inhibition appeared to affect most cells exposed and was equally effective against glycoprotein and protein synthesis. The inhibition of protein metabolism was independent of mRNA synthesis and uptake of labelled precursors into intracellular pools, indicating that it was directed at intracellular translational events. Fractionation of chloroform/methanol-extracted preparations of this brain cell-surface substance on Bio-Gel P-100 revealed the material to be quite heterogenous, although inhibitory activity was found only in fractions of mol.wt. 25000—30000 and 6000—10000. Biochemical analysis of these fractions demonstrated that they were 6% carbohydrate and 94% amino acid by weight. The 25000—30000-mol.wt. glycopeptides were shown to inhibit cell growth at concentrations of 2 microgram/ml in cultured cells and to inhibit protein synthesis by 50% at concentrations of 3 microgram/ml. The 25000—30000-mol.wt. brain-cell-surface-substance glycopeptides were further purified by ultrafiltration and affinity chromatography with Ulex europaeus agglutinin, resulting in a 400-fold increase in specific biological activity. The inhibitor was not lethal to cells and was not species- or tissue-specific.
Permeability of lactating-rat mammary gland Golgi membranes to monosaccharides
Authors: White M D , Kuhn N J , Ward S
Published: August 2015

The Golgi-membrane vesicles present in particulate preparations of lactating rat mammary gland were biosynthetically loaded with [14C]lactose. This lactose was effectively retained by particles sedimented after exposure to 0.25 M-disaccharide, but was partly lost after exposure to 0.25 M-glucose or other solutes of similar size. Loss of lactose was time-, concentration- and temperature-dependent and varied with the solute structure. This behaviour is ascribed to the presence of protein in the Golgi membrane, forming a specific carrier or channel that serves to supply glucose for lactose synthesis.
Changes in the surface membrane during myoblast fusion
Authors: Curtis D H , Micklem K J , Gill K , Pasternak C A
Published: August 2015

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.
Organic-acid transport in resealed haemoglobin-containing human erythrocyte ‘ghosts’
Authors: Hubbard A R , Sprandel U , Chalmers R A
Published: August 2015

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte ‘ghosts’ prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte ‘ghosts’ retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte ‘ghosts’ from patients with hyperoxaluria was also studied.
Transcriptionally active RNA polymerases from Morris hepatomas and rat liver. Elucidation of the mechanism for the preferential increase in the tumour RNA polymerase I
Authors: Duceman B W , Jacob S T
Published: August 2015

The amount and/or activity of DNA-dependent RNA polymerase I, Ii and III from resting liver, regenerating liver and a series of Morris hepatomas (5123D, 7800, 7777, 3924A) were determined after extraction of the enzymes from whole tissue homogenates and subsequent fractionation by DEAE-Sephadex column chromatography. When compared with resting liver, the tumours exhibited a characteristic enzyme pattern in which polymerase I, but not II, was increased. The increase in RNA polymerase I was proportional to the tumour growth rates. Alterations in polymerase III were confined to the most rapidly proliferating hepatomas. By contrast, all classes of RNA polymerase were found to be increased during liver regeneration. Relative to resting liver, the fastest growing tumour, 3924A, exhibited the highest activities and/or amounts of RNA polymerase I (8-fold) and III (5-fold) per g of tissue. These alterations in the tumour RNA polymerases were reflected in corresponding increases in the transcriptionally active (bound or chromatin-associated) enzyme population. The mechanisms underlying the augmented synthesis of RNA in vitro by bound polymerase I from hepatoma 3924A were elucidated by product analysis. The results indicated that, relative to liver RNA polymerase I, the tumour enzyme produced more nascent RNA chains and elongated these chains at a faster rate. The number of 3'-termini, as measured by incorporation into uridine, was higher in the hepatoma even under conditions which prevented re-initiation. suggesting increased amount of transcriptionally active RNA polymerase I in the tumour.
Spectrophotometric assay, solubilization and purification of brain 2′:3′-cyclic nucleotide 3′-phosphodiesterase
Authors: Nishizawa Y , Kurihara T , Takahashi Y
Published: August 2015

1. A spectrophotometric assay of 2′:3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) based on the use of an acid-base indicator and a buffer having identical pKa values is described. The assay is simple and rapid; it was particularly convenient for monitoring the enzyme activity at various stages of purification. 2. Several proteinases were examined for their ability to solubilize 2′:3′-cyclic nucleotide 3′-phosphodiesterase from delipidated brain white matter. Trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.11) appeared to be more effective than the other proteinases examined. Trypsin, however, caused inactivation; elastase was therefore chosen to solubilize 2′:3′-cyclic nucleotide 3′-phosphodiesterase. When a partially purified preparation of 2′:3′-cyclic nucleotide 3′-phosphodiesterase was treated with elastase, 2′:3′-cyclic nucleotide 3′-phosphodiesterase was solubilized nearly quantitatively. Elastatinal, a specific inhibitor of elastase, specifically inhibited the solubilization with elastase. 3. 2′:3′-cyclic nucleotide 3′-phosphodiesterase was purified from bovine brain white matter by: (i) delipidation; (ii) solubilization with hexadecyltrimethylammonium bromide; (iii) gel chromatography on Sepharose; (iv) ethanol precipitation and resolubilization by digestion with elastase; (v) chromatography on DEAE-Sephadex; (vi) affinity chromatography on 8-(6-aminohexyl)amino-2′-AMP-Sepharose. 4. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the estimated mol.wt. in the latter electrophoresis was 27000-31000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a mol.wt. of 31000. Therefore the purified enzyme is a monomer protein with a mol.wt. of approx. 30000.
Production of a variant of β-lactamase II with selectively decreased cephalosporinase activity by a mutant of Bacillus cereus 569/H/9
Authors: Baldwin G S , Edwards G F S , Kiener P A , Tully M J , Waley S G , Abraham E P
Published: August 2015

1. Mutants of Bacillus cereus 569/H/9 have been screened in a search for strains that synthesize variants of beta-lactamase II. 2. One of these mutants (strain 569/H/9/1) produces a beta-lactamase II-like enzyme that shows a selective decrease in cephalosporinase activity. 3. beta-Lactamase II from strain 569/H/9/1 has been purified to apparent homogeneity and its kinetic properties have been examined. This enzyme resembles the parent beta-lactamase II in its relative activity with benzylpenicillin as substrate when Zn(II) is replaced by other metal ions, but differs detectably from the parent enzyme in its isoelectric point.
Identification of the subunits of bovine heart mitochondrial NADH dehydrogenase that are exposed to the phospholipid bilayer by photo-labelling with 5-iodonaphth-1-yl azide
Authors: Earley F G P , Ragan C I
Published: August 2015

Mitochondrial NADH dehydrogenase may be isolated from bovine heart as a lipoprotein complex (Complex I or NADH-ubiquinone oxidoreductase). Polypeptide subunits that are exposed to the hydrophobic region of the phospholipid bilayer were identified by photolabelling with the hydrophobic probe, 5-[125I]iodonaphth-1-yl azide. Chaotropic resolution of the labelled enzyme showed that the hydrophilic flavoprotein and iron-protein fragments of the enzyme were not in contact with the phospholipid bilayer. When complex I that had been partially depleted of phospholipids was photolabelled, incorporation of radioactivity into certain polypeptides was increased, indicating either conformational changes in protein or preferential association of these polypeptides with residual cardiolipin. A model NADH dehydrogenase structure is proposed on the basis of these results and those obtained with hydrophilic probes by Smith & Ragan (1980) Biochem. J. 185, 315-326.
Antigenic determinants of a plant proteoglycan, the Gladiolus style arabinogalactan-protein
Authors: Gleeson P A , Clarke A E
Published: August 2015

Antiserum has been raised to the arabinogalactan-protein of Gladiolus style mucilage. This macromolecule has been characterized and has a structure consistent with a 1 leads to 3-linked beta-galactan backbone with side branches of 1 leads to 6-linked beta-galactosyl residues, some of which carry terminal alpha-L-arabinofuranoside residues [Gleeson & Clarke (1979) Biochem. J. 181, 607-621]. The specificity of the antiserum has been investigated by immunoprecipitation with [3H]arabinogalactan-protein. THe 3H label was introduced into the arabinogalactan-protein by oxidation of the terminal galactose residues with galactose oxidase, followed by reduction with NaB3H4. The antigenic specificity of the antiserum was shown to be directed towards the carbohydrate component of the arabinogalactan-protein. D-galactose and L-arabinose were the most effective hapten inhibitors of the antiserum; other monosaccharides, N-acetyl-D-galactono-1,4-lactone, D-glucose, D-mannose, L-rhamnose. L-fucose and D-xylose, were all poor inhibitors. The antiserum showed preference for beta-galactosides over alpha-galactosides. Of the haptens examined, the disaccharide 6-O-beta-D-galactopyranosyl-D-galactopyranose was the most potent inhibitor. The antigenic features of the arabinogalactan-protein were investigated by examining the interaction of the antiserum with chemically and enzymically modified arabinogalactan-protein. Also, the cross-reactivity of structurally related polysaccharides and glycoproteins with the specific antiserum was assessed by a haemagglutination assay using erythrocytes coupled with specific antiserum. The results indicate that the dominant antigenic determinants of the arabinogalactan-protein are probably the side branches of 1 leads to 6 -linked beta-galactose residues bearing the terminal alpha-L-arabinose residues.
A Mössbauer spectroscopic investigation of the redox behaviour of the molybdenum-iron protein from Klebsiella pneumoniae nitrogenase. Mechanistic and structural implications
Authors: Smith B E , O'Donnell M J , Lang G , Spartalian K
Published: August 2015

The redox properties of the nitrogenase Mo-Fe protein from Klebsiella pneumoniae have been monitored by 57Fe Mössbauer spectroscopy between -460 and -160mV (relative to the normal hydrogen electrode). Two redox processes associated with the atoms of the protein were observed. One at -216mV (pH 8.7) was associated with the Fe-Mo cofactor centres in the protein and allowed identification of the Mössbauer parameters of the oxidized form of these centres. The other redox process at -340mV (pH 8.7) was associated with species M5 [Smith & Lang (1974) Biochem. J. 137, 169-180]. This latter redox process may be involved in enzyme turnover. The oxidized form of species M5 interacts magnetically with species M4. The structural implications of the data have been considered in relation to other published data. It is concluded that an unequivocal assignment of the M4 and M5 atoms to Fe-S cluster types is not yet possible.
Further studies on the substrate specificity and inhibition of the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena
Authors: Barrett Carol H. , Dodgson Kenneth S. , White Graham F.
Published: August 2015

A series of d-alkan-2-yl sulphate esters (C7–C14) were prepared by sulphation of the resolved parent alcohols by a method that entails complete retention of configuration. These sulphate esters were tested as substrates for the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena. Vmax. reached a maximum with the C9 compound, whereas logKm decreased linearly as the alkyl-chain length was increased from C7 to C14. A parallel series of l-alkan-2-yl sulphates was also prepared, and these esters, together with homologous series of primary alkyl sulphates and primary alkanesulphonates, were shown to be competitive inhibitors of the CS2 enzyme. For each series of compounds, logKi values decreased linearly with increasing alkyl-chain length. Plots of chain length against the standard free energy of binding (ΔG0) of substrate and inhibitors to the CS2 enzyme showed that the standard free energy of association of a –CH2– group with the enzyme was 2.0–2.4kJ/mol for all classes of compound studied, indicating an important contribution from hydrophobic interactions to the overall binding. Plots for d-alkan-2-yl sulphate substrates and primary alkyl sulphate inhibitors were nearly coincident, suggesting that the overall interaction between a primary ester and the enzyme is the same as that between the isomeric secondary substrate and the enzyme. Plots for l-alkan-2-yl sulphate and alkanesulphonate inhibitors were very similar to each other, but were displaced by 1.5–3.0kJ/mol from that for substrate binding. This indicates that the binding of any one of these particular inhibitors involves one carbon atom fewer than the number involved in binding a substrate of the same chain length. These observations are discussed in terms of a three-point attachment of substrate to the enzyme involving the alkyl chain, sulphate group and the C-1 methyl group.
Complex lipids of a lipolytic and general-fatty-acid-requiring Butyrivibrio sp. isolated from the ovine rumen
Authors: Hazlewood G P , Clarke N G , Dawson R M C
Published: August 2015

The complex lipids of the naturally-occurring general-fatty-acid-auxotroph Butyrivibrio S2 [Hazlewood & Dawson (1979) J. Gen. Microbiol. 112, 15-27] grown with palmitic acid as sole fatty-acid supplement have been investigated and some have been isolated in a state of purity and analysed. The majority are phospholipids (84%) and many contain galactose. They typically possess few esterified long-chain fatty-acid residues (C16:0), but are rich in esterified butyric acid and C16-alkenyl groups. Most of the phosphorus-containing lipids, including the two major lipids of the organism, contain esterified diabolic acid, a long-chain vicinal dimethyl-substituted dicarboxylic acid [Klein, Hazlewood, Kemp & Dawson (1979) Biochem. J. 183, 691-700] in definite stoichiometric relationship to phosphorus. No phosphatidylglycerol was present, but its monobutyroyl ester was detected as a minor component. Galactofuranosyldiacylglycerol (plasmalogen) and its monobutyroyl ester, cetyl alcohol and diacylglycerol were also identified.
The interpretation of entropy/enthalpy compensation phenomena in the deacylation of acyl-α-chymotrypsins
Authors: Adams P A
Published: August 2015

The thermodynamic-compensation law observed for the deacylation of a series of acyl-alpha-chymotrypsins has been re-examined. From consideration of the effect of small changes in delta delta H+(+)delta T along a homologous series, it is suggested that the high Tc value of 420 K observed for the process has its origin in the solvation of the acyl-group-catalyst transition state.
Characterization of collagenous meshworks by volume exclusion of dextrans
Authors: Bert J L , Pearce R H , Mathieson J M , Warner S J
Published: August 2015

The volumes from which 3H-labelled dextrans are excluded by dermal collagenous fibres were calculated by dilution of dextran probes. Five dextrans, of average Stokes' radii 1.72, 2.53, 3.92, 4.54 and 14.24nm, were investigated at concentrations between 0.1 and 3% (w/w). The excluded volume was dependent on dextran concentration only for the two smaller probes. The largest dextran was shown not to bind to the fibres. A plot of the square root of excluded volume against Stokes' radius was linear for the four smallest dextrans, corresponding to the predictions of Ogston's [(1958) Trans. Faraday Soc. 54, 1754–1757] rod-and-sphere model of fibrous exclusion, and suggesting that dextrans of Stokes' radius between 1.72 and 4.54 nm were excluded by a cylindrical solid fibre of radius 2.90 +/- 0.72 nm. Larger molecules were excluded by a structure of much greater size, since the volume exclusion for the largest dextran was only slightly greater than that of the dextran less than one-third its radius. The excluded volume of 3H2O fell slightly below the line describing the dextran data, indicating that water had access to most of the volume not occupied by the collagenous fibres.
Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin
Authors: Sutcliffe R G , Kukulska-Langlands B M , Coggins J R , Hunter J B , Gore C H
Published: August 2015

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.
Immobilization of DNA for affinity chromatography and drug-binding studies
Authors: Macdougall A J , Brown J R , Plumbridge T W
Published: August 2015

A method is described in which double-stranded DNA is alkylated with 4-bis-(2-chloroethyl)amino-L-phenylalanine and the product immobilized on an insoluble support via the primary amino group of the phenylalanine moiety. The DNA is hence irreversibly bound to the matrix by both strands at a limited number of points.
Pregnancy-related changes in rat cervical glycosaminoglycans
Authors: Golichowski A M , King S R , Mascaro K
Published: August 2015

Non-pregnant and pregnant rats of known gestational age were killed at intervals and their uterine cervices were excised and digested with papain. Glycosaminoglycans thus extracted were separated by cellulose acetate electrophoresis and stained with Alcian Blue. Glycosaminoglycans were identified by comparison with standards and by serial degradation with chondroitin ABC lyase, butyl nitrite and leech hyaluronidase. Dermatan sulphate, hyaluronic acid and heparan sulphate were identified and quantitative determined by densitometry. The overall concentration of glycosaminoglycans changed little during pregnancy. A 3-fold total increase in uronic acid paralleled the increase in cervical weight. Hyaluronate content, however, increased 17-fold, and rose from 6% of total glycosaminoglycans in the non-pregnant state to 33% at term. Furthermore, the ratio of hyaluronate to hydroxyproline increased 10-fold. These changes are consistent with an accumulation of hyaluronate in the interstices between collagen fibres, resulting in the softening of this tissue that is seen in late pregnancy.
The reconstitution of l-3-glycerophosphate-cytochrome c oxidoreductase from l-3-glycerophosphate dehydrogenase, ubiquinone-10 and ubiquinol—cytochrome c oxidoreductase
Authors: Cottingham I R , Ragan C I
Published: August 2015

Purified L-3-glycerophosphate dehydrogenase from pig brain mitochondria interacts with ubiquinone-10 and ubiquinol-cytochrome c oxidoreductase (Complex III) from bovine heart mitochondria to reconstitute antimycin-sensitive L-3-glycerophosphate- cytochrome c oxidoreductase. This activity is completely dependent on the two enzymes and largely dependent on ubiquinone-10. Reconstitution requires that the two enzymes should be simultaneously present in the same membranous aggregate produced by removal of detergent from the enzymes. Reconstitution by removing detergent by dialysis or dilution is inefficient because of self-aggregation of the dehydrogenase. Highly efficient reconstitution can be achieved if the enzymes are co-precipitated by addition of ethanol. The rate with reconstituted enzyme approaches that expected from the turnover of the dehydrogenase with ubiquinone-1 as acceptor. The behaviour of the reconstituted system shows some of the characteristics expected for a stoicheiometric association of one molecule of dehydrogenase with one molecule of Complex III. On raising the phospholipid/protein ratio, the dehydrogenase and Complex III appear to operate as independent enzymes acting in sequence. These effects are very similar to those observed for the interaction of NADH dehydrogenase and Complex III and are explained in terms of the model proposed by Heron, Ragan & Trumpower [(1978) biochem. J. 174, 791-800].
Biochemical changes during reticulocyte maturation in culture. A comparison of genetically different sheep erythrocytes
Authors: Tucker E M , Young J D
Published: August 2015

A simple culture technique was used to follow sheep reticulocyte maturation. Cells remained in good condition without lysis for at least 7 days and maintained normal intracellular ATP concentrations. Amino acid and nucleoside transport were measured during maturation together with changes in reduced glutathione (GSH), amino acid, K+ and Na+ concentrations. Reticulocytes of the genetic types high-K+ and low-K+, high-GSH and low-GSH; nucleoside-permeable and nucleoside-impermeable were indistinguishable both in transport properties and cellular constituents. Cell maturation was associated with marked decreases in L-alanine transport (Na+-dependent and independent), uridine transport and GSH and K+ concentrations. Typical mature-cell levels for all parameters were reached within the culture period, during which time predicted differences between cells of different genotypes became apparent. Loss of amino acid-transport activity from low-GSH cells was accompanied by an increase in intracellular ornithine and lysine concentrations. The rates at which different parameters changed during culture were compared.
Effects of phenobarbital upon triacylglycerol metabolism in the rabbit
Authors: Goldberg D M , Roomi M W , Yu A , Roncari D A K
Published: August 2015

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.
Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31 phosphorus nuclear magnetic resonance study
Authors: Iles R A , Griffiths J R , Stevens A N , Gadian D G , Porteous R
Published: August 2015

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.
Phosphatidylinositol phosphodiesterase in higher plants
Authors: Irvine R F , Letcher A J , Dawson R M C
Published: August 2015

1. The lower regions of the stem of celery (Apium graveolens L.) contain a soluble enzyme that hydrolyses phosphatidylinositol. 2. The lipoidal product of hydrolysis is diacylglycerol, and the water-soluble products are 1:2-cyclic phosphoinositol and phosphoinositol in the approximate proportions of 60% and 40% respectively: this indicates that a phosphodiesterase (phospholipase C-like) activity is cleaving the phosphatidylinositol. 3. The enzyme requires a bivalent cation, Ca2+ being the most effective activator. 4. The enzyme has a pH optimum, depending on conditions of assay, of pH 5.9-6.6 and in this pH range shows no detectable activity against phosphatidylcholine or phosphatidylethanolamine. 5. The activity is stimulated by phosphatidic acid and slightly inhibited (30% at concentrations equimolar with phosphatidylinositol) by phosphatidylcholine. 6. The phosphodiesterase was also detected (but not quantified) in the tips of the flowers in cauliflowers, in outer leaves of onion and in the elongating stem of daffodils. 7. The enzyme's properties are compared with equivalent mammalian enzymes, and its possible role in the catabolism of phosphatidylinositol in higher plants is discussed.
Effect of glucose on carbohydrate synthesis from alanine or lactate in hepatocytes from starved rats
Authors: Solanki K , Nyfeler F , Moser U K , Walter P
Published: August 2015

In hepatocytes from starved rats, 10mM-glucose suppressed in incorporation of 2mM labelled alanine into glucose+glycogen by more than 40%, whereas no inhibition was observed with labelled lactate as substrate. Addition of glycerol instead of glucose did not show this inhibition. The inhibitory effect could also be demonstrated in label-free experiments.
Correlation of nuclear acceptor sites for oestrogen receptors with gene transcription in vitro
Authors: Taylor Robert N. , Swaneck George E. , Smith Roy G.
Published: August 2015

The interaction of oestrogen receptors with their nuclear acceptor sites was studied to ascertain whether these acceptor sites are involved in the regulation of ovalbumin-gene expression in the chick oviduct. As previously described, two distinct oestrogen-receptor species exist, and both are translocated into the nucleus after oestrogen administration in vivo [Smith, Clarke, Zalta & Taylor (1979) J. Steroid Biochem.10, 31–35]. In the present investigation we observed that the tubular-gland-cell concentrations of cytoplasmic receptors (800–900/cell) do not vary with prolonged withdrawal, nor do the relative ratios of the two receptor types change; however, the nuclear accumulation and retention of these receptors after secondary oestrogen administration are attenuated in a time-dependent fashion. Chicks were withdrawn from oestrogen for 24–84h. Some animals were then restimulated with oestrogen and killed after 4h, when oviduct nuclei were isolated. These nuclei were assayed for nuclear receptor concentrations and for their capacity to synthesize ovalbumin mRNA in vitro. Although an equal number of cytoplasmic receptors appeared to be translocated, oestrogen-receptor occupancy within the nucleus was not equal, but was inversely proportional to the preceding length of withdrawal. This decrease in nuclear acceptor sites was accompanied by a similar decrease in the capacity of these same nuclei to transcribe ovalbumin mRNA in vitro. A statistical evaluation of nuclear oestrogen-receptor concentrations and ovalbumin-mRNA synthesis in vitro was made. Correlation analysis revealed a Pearson coefficient r=0.87 (P<0.001, n=17), indicating that a high degree of correlation exists between these two parameters. These results are consistent with the hypothesis that nuclear oestrogen-receptor-acceptor complexes may correspond to initiation sites for RNA polymerase II transcription of an oestrogen-regulated gene.
Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture
Authors: Sudhakaran Perumana R. , Sinn Wolfgang , von Figura Kurt
Published: August 2015

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E1, and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of 35SO42− into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of 35SO42− or [14C]glucosamine into heparan sulphate was decreased to 40–50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse–chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by β-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, 35SO42− incorporation was decreased to 5%. Addition of p-nitrophenyl β-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl β-d-xylosides. Incorporation of [3H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.
The inhibitory action of l-dihydroxyphenylalanine (l-dopa) in the presence of a monoamine oxidase inhibitor on protein synthesis in vivo and the partial amelioration of this by methionine supplementation
Authors: Bone A H , Taufek H R
Published: August 2015

Male Wistar rats of various age groups were injected daily over a period of 3 weeks with iproniazid (10 micrograms/g body wt.) and L-dihydroxyphenylalanine (L-dopa; 0.1 mg/g body wt.). On the final day 1 h before the termination of the experiment the animals were injected with L-[14C]valine (0.1 microCi/g body wt.). The specific radioactivity of the valine in the proteins of the subcellular fractions of the tissues examined, relative to the time-integrated mean specific radioactivity of this amino acid in the acid-soluble pools of these tissues, was used to assess protein synthesis. The L-dopa/monoamine oxidase-inhibitor treatment was associated with 30–40% inhibition of protein synthesis. Supplementation of the dietary methionine intake by injection of this amino acid markedly diminished the inhibitory action of the L-dopa/monoamine oxidase-inhibitor treatment on protein synthesis in all fractions examined.
Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle
Authors: Akhtar Rashid A. , Abdel-Latif Ata A.
Published: August 2015

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca2+ was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50μm) increased the breakdown of phosphatidylinositol bisphosphate in [3H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of 3H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the 3H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20μm), but not tubocurarine (100μm); and it was abolished by depletion of extracellular Ca2+ with EGTA, but restored on addition of low concentrations of Ca2+ (20μm). 5. Calcium-antagonistic agents, such as verapamil (20μm), dibenamine (20μm) or La3+ (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (‘microsomes’) was stimulated by 43% in the presence of 50μm-Ca2+. 7. The results indicate that increased Ca2+ influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca2+ activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the 3H radioactivity of this phospholipid.
On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate
Authors: Glaser G , Giloh H , Kasir J , Gross M , Mager J
Published: August 2015

Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution (“starved cells”) resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.
Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential
Authors: Scott I D , Akerman K E , Nicholls D G
Published: August 2015

The association of Ca2+ with isolated nerve endings (synaptosomes) is investigated and resolved into two components, that bound to the outer surface of the plasma membrane and that transported across the plasma membrane. When synaptosomes are added directly to a Ca2+-containing medium, there is an initial rapid uptake of Ca2+ across the plasma membrane, followed by a slow uptake that proceeds for 20 min. The rapid phase is not observed if the synaptosomes are initially pre-incubated in a Ca2+-free medium. Rapid disruption of synaptosomes reveals that less than 3 nmol of transported Ca2+ per mg of synaptosomal protein can be ascribed to non-mitochondrial components, whereas the remainder, up to 79% of the total, is further transported into the mitochondrial matrix. Abolition of oxidative phosphorylation while the mitochondrial membrane potential is retained leads to a time-dependent increase in transported Ca2+, whereas abolition of the mitochondrial membrane potential decreases both plasma-membrane transport and accumulation of Ca2+ in the mitochondrial matrix. It is concluded that intrasynaptosomal mitochondria are major regulators of synaptosomal Ca2+.
Is regulation of proteolysis associated with redox-state changes in rat skeletal muscle?
Authors: Tischler M E
Published: August 2015

In isolated rat diaphragms, only those substrates that increased the tissue NADH/NAD+ ratio lowered the rate of proteolysis. However, direct inhibition of proteinase activity by leupeptin promoted oxidation of the NAD couple of the muscles. These results suggest that changes in muscle reduction-oxidation state may be important in the regulation of proteolysis.