bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D)
Authors: Findlay D M , Michelangeli V P , Moseley J M , Martin T J
Published: August 2015

Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines.
Peroxisomal fatty acid oxidation as detected by H2O2 production in intact perfused rat liver
Authors: Foerster E C , Fährenkemper T , Rabe U , Graf P , Sies H
Published: August 2015

1. H2O2 formation associated with the metabolism of added fatty acids was quantitatively determined in isolated haemoglobin-free perfused rat liver (non-recirculating system) by two different methods. 2. Organ spectrophotometry of catalase Compound I [Sies & Chance (1970) FEBS Lett. 11, 172-176] was used to detect H2O2 formation (a) by steady-state titration with added hydrogen donor, methanol or (b) by comparison of fatty-acid responses with those of the calibration compound, urate. 3. In the use of the peroxidatic reaction of catalase, [14C]methanol was added as hydrogen donor at an optimal concentration of 1 mM in the presence of 0.2 mM-L-methionine, and 14CO2 production rates were determined. 4. Results obtained by the different methods were similar. 5. The yield of H2O2 formation, expressed as the rate of H2O2 formation in relation to the rate of fatty-acid supply, was less than 1.0 in all cases, indicating that, regardless of chain length, less than one acetyl unit was formed per mol of added fatty acid by the peroxisomal system. In particular, the standard substrate used with isolated peroxisomal preparations (C16:0 fatty acid) gave low yield (close to zero). Long-chain monounsaturated fatty acids exhibit a relatively high yield of H2O2 formation. 6. The hypolipidaemic agent bezafibrate led to slightly increased yields for most of the acids tested, but the yield with oleate was decreased to one-half the original yield. 7. It is concluded that in the intact isolated perfused rat liver the assayable capacity for peroxisomal beta-oxidation is used to only a minor degree. However, the observed rates of H2O2 production with fatty acids can account for a considerable share of the endogenous H2O2 production found in the intact animal.
Brain 5-aminolaevulinate synthase Developmental aspects and evidence for regulatory role
Authors: De Matteis F , Zetterlund P , Wetterberg L
Published: August 2015

1. Brain 5-aminolaevulinate synthase showed a peak of increased activity in the first few weeks of life, which preceded and accompanied the development of brain cytochromes. 2. In the brain of the adult rat the activity of the enzyme was only 20% of that in the liver (on a per g wet wt. basis), but it was still probably sufficient to maintain the turnover of brain cytochromes. 3. The brain synthase activity could be decreased by treatment of rats with cycloheximide or with large doses of 5-aminolaevulinate, especially when this precursor was given as the methyl ester. 4. Injected haematin and CoCl2 markedly inhibited the synthase activity in the liver but failed to affect the brain enzyme; neither was taken up by the brain in vivo. 5. It is concluded that the brain can itself produce the haem required for the synthesis and turnover of its own haemoproteins and that 5-aminolaevulinate synthase may regulate the pathway in brain as in other tissues. 6. The relevance of the present findings to the pathogenesis of the neurological symptoms of acute porphyria and to the beneficial effect of exogenous haematin in porphyric patients is briefly considered.
Mitochondrial-membrane polar-head-group composition is influenced by diet fat
Authors: Innis S M , Clandinin M T
Published: August 2015

Male Sprague-Dawley rats were fed diets containing 20% (w/w) soya-bean oil, high-erucic acid rapeseed oil or low-erucic acid rapeseed oil for 0, 12 or 23 days. The type of fat present in the diet had no effect on the total phospholipid content of heart mitochondria (micrograms/mg of protein) but did influence the phospholipid class distribution. Rats fed high-erucic acid rapeseed oil for 12 or 23 days had significantly higher mitochondrial phosphatidylcholine content than rats fed soya-bean oil. Low-erucic acid rapeseed oil resulted in elevation of cardiac mitochondrial cardiolipin content after dietary treatment for 12 days. The results demonstrate in vivo that diet is a significant determinant of the phospholipid class content of subcellular membranes.
Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro
Authors: King Ian A. , Tabiowo Anne
Published: August 2015

1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[3H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-14C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[3H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[3H]glucosamine but not of (U-14C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[3H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[3H]glucosamine and 35SO42− into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.
The effect of essential-fatty-acid deficiency on the activity of liver phosphatidate phosphatase in rats
Authors: Stewart James H. , Briggs George M.
Published: August 2015

The activities of the soluble and microsomal phosphatidate phosphatases (EC 3.1.3.4) are greater in the livers of essential-fatty-acid-deficient rats than in rats fed diets containing linoleic acid.
Determination of glutathione in lymphocytes and possible association of redox state and proliferative capacity of lymphocytes
Authors: Noelle R J , Lawrence D A
Published: August 2015

The glutathione (GSH) content of mouse T- and B-cells was determined and compared with the GSH content of human peripheral blood lymphocytes and human erythrocytes. Owing to the difficulty of obtaining large numbers of purified lymphocytes, a technique was developed to measure picomolar quantities of GSH. By this technique, mouse T- and B-cells, as well as mouse peripheral-blood lymphocytes, were found to contain approx. 30% of the GSH found in human peripheral-blood lymphocytes. The concanavalin A response of human peripheral-blood lymphocytes and human spleen cells was insensitive to 2-mercaptoethanol as well as to culture in 17% O2, whereas mouse lymphocyte responses were altered by 2-mercaptoethanol and inhibited by 17% O2. The capacity of human peripheral-blood lymphocytes, human erythrocytes, mouse T-cells and mouse B-cells to regenerate GSH stores after chemical oxidation by diamide was tested, and it was found that mouse cells were less capable of regenerating GSH than human erythrocytes or human peripheral-blood lymphocytes. In addition, the latter lymphocytes were less sensitive to oxidation of GSH and to inhibition of proliferation by diamide.
The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography
Authors: Wilson K J , Honegger A , Stötzel R P , Hughes G J
Published: August 2015

High-pressure (‘performance’) liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.
A generalized theory of the transition time for sequential enzyme reactions
Authors: Easterby J S
Published: August 2015

In a sequence of coupled enzyme reactions the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. Provided that a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaelis-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Application of the theory to coupled enzyme assays allows a definition of the minimum requirements for successful operation of the assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration.
The amino acid sequence and position of the free thiol group of a short-chain neurotoxin from common-death-adder (Acanthophis antarcticus) venom
Authors: Kim H S , Tamiya N
Published: August 2015

The amino acid sequence of a short-chain neurotoxin Acanthophis antarcticus c (toxin Aa c) from the venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus, subfamily Acanthophiinae) was elucidated. Toxin Aa c is composed of 62 amino acid residues, including eight half-cystine residues and a cysteine residue. The amino acid sequence of toxin Aa c is homologous with those of other short-chain neurotoxins found in snakes of the family Elapidae, especially with those from snakes of the subfamily Hydrophiinae. The single cysteine residue was located in position 4. Toxin Aa c has a lethal dose (LD50) of 0.08 micrograms/g body weight of mouse on intramuscular injection.
Purification and properties of the methanol dehydrogenase from Methylophilus methylotrophus
Authors: Ghosh R , Quayle J R
Published: August 2015

1. A dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown Methylophilus methylotrophus. 2. The enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees C. 3. The value of A1% 1cm,280 was 17.5. 4. The enzyme retained bound methanol after passage through Sephadex G-25. This tightly-bound methanol slowly exchanged with free [14C]-methanol from a value of 0.27 mol of [14C]methanol/mol of enzyme after 48 h incubation at 4 degrees C to a limiting value of approx. 2.5 mol of [14C]methanol/mol of enzyme after 3 weeks incubation at 4 degrees C. 5. One mol of this enzyme reduced 89.4 mol of 2,6-dichlorophenol-indophenol (via phenazine methosulphate) in the absence of any additional methanol in the assay mixture. The source of the electrons involved in this reduction is not known.
A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronate-substituted Sepharose gel
Authors: Tengblad A
Published: August 2015

The hyaluronate-binding proteins from bovine nasal cartilage, i.e. the hyaluronate-binding region of the proteoglycan and the link protein, were labelled with 125I and separated from each other by gel chromatography. The proteins were characterized by molecular-weight determinations and their purity was established by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and immunodiffusion. The binding properties of the two proteins by hyaluronate-substituted Sepharose gel were compared. It was found that both proteins behaved similarly. They bound with the same efficiency to the gel, they showed the same time course of binding, had slightly different pH optima for binding and both proteins had a decreasing affinity for the gel with increasing ionic strength. The binding to the gel could be inhibited by soluble hyaluronate, and the minimum size of a hyaluronate oligosaccharide required for inhibition was in both cases a decasaccharide (only even-numbered oligosaccharides were tested). The proteins did not show any co-operative binding in the system tested, which could be explained by the large number of binding sites in the hyaluronate-substituted gel. Binding constants for the protein-hyaluronate interaction were estimated. A value of 1.3 x 10(7) M-1 was obtained for the hyaluronate-binding region of the proteoglycan, in agreement with literature data. The corresponding value for the link protein was 0.7 x 10(7) M-1.
Kinetic and physicochemical characteristics of an endogenous inhibitor to progesterone–receptor binding in rat placental cytosol
Authors: Ogle T F
Published: August 2015

This study describes the kinetic behaviour and physicochemical aspects of an endogenous inhibitor of progesterone--receptor binding in trophoblast cytosol from day-12 embryos. The progesterone cytosol receptor was partially purified and isolated from the inhibitor as the 0--50%-satd. (NH4)2SO4 fraction. The inhibitory substance was shown to reside in the 50--70%-satd. (NH4)2SO4 fraction. Equilibration of the inhibitor preparation with the receptor fraction increased the Kapp.D of the ligand--receptor binding reaction in a concentration-dependent manner (26 +/- 3-fold increase in Kapp.D per mg of protein of the (NH4)2SO4 fraction, n = 16). However, the inhibitor did not alter the concentration of binding sites. Studies of other physicochemical aspects of the inhibitor showed it to be non-diffusible, excluded from Sephadex G-25, stable at 35 degrees C for 30 min, but irreversibly denatured at 70 degrees C for 30 min. The Stokes′ radius was estimated by gel chromatography to be 2.8 +/- 0.11 nm (n = 5). Inhibitory activity was destroyed by HgCl2, suggesting that disulphide bridges play an essential role in the biological activity of this molecule. The inhibitor is a macromolecule which does not bind progesterone and differs from albumin. The kinetic mechanism by which the inhibitor enhanced Kapp.D was investigated by measuring association and dissociation rate constants and the energy of activation (Ea) for each reaction. The association rate (k+1) for progesterone and receptor was (1.3 +/- 0.2) x 10(4) M-1 . s-1 but declined to (0.4 +/- 0.1) x 10(4) M-1 . s-1 (n = 5) when exposed to the inhibitor (P less than 0.01). The dissociation rate (k-1) was (3.2 +/- 0.6) x 10(-5) s-1 for progesterone--receptor complex and was unchanged by the inhibitor. The Ea for the association of complex was 33.6 +/- 4.2 kJ/mol and was increased to 63.0 +/- 8.4 kJ/mol by the inhibitor (P less than 0.05). The Ea of dissociation was unaltered. Thus, an inhibitor is present in trophoblast cytosol which specifically enhances Kapp.D without altering availability of binding sites. The mode of action of inhibitor is to increase the energy of activation for association of complex without influencing the dissociation reaction.
Design, synthesis and DNA-binding capacity of a new peptidic bifunctional intercalating agent
Authors: Bernier J L , Henichart J P , Catteau J P
Published: August 2015

A lysyl-lysine bifunctional derivative of 9-aminoacridine has been synthesized and its DNA-binding capacity established by electron-paramagnetic-resonance study. For this purpose the binding parameters of a spin-labelled aminoacridine probe were estimated and the affinities of the lysylacridinyl-lysyldiamino-octane dimer and of 9-amino-acridine could be evaluated by competitive assays. The competition study provided quantitative results concerning the dissociation constant (KD) of the dimer. The obtained value was closely similar to the KD of 9-aminoacridine determined by the same method and to the KD previously reported for the anti-tumour and antibiotic bifunctional intercalator quinomycins.
N-terminal sequence analysis of polypeptide at the picomole level
Authors: Chang J Y
Published: August 2015

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.
Coupling of [33S]sulphur to molybdenum(V) in different reduced forms of xanthine oxidase
Authors: Malthouse J P G , George G N , Lowe D J , Bray R C
Published: August 2015

Different reduced forms of xanthine oxidase, labelled specifically in the cyanide-labile site with 33S, were prepared and examined by electron paramagnetic resonance. Coupling of this isotope to molybdenum(V) was quantified with the help of computer simulations and found to differ markedly from one reduced form to another. The xanthine Very Rapid signal shows strong, highly anisotropic, coupling with A(33S)av. 1.27 mT. For this signal, axes of the g- and A(33S)-tensors are rotated relative to one another. One axis of the A-tensor is in the plane of gxx ang gyy, but rotated by 40 degrees relative to the gxx axis, whereas the direction of weakest coupling to sulphur deviates by 10 degrees from the gzz axis. In contrast with this signal, only rather weaker coupling was observed in different types of Rapid signal [A(33S)av. 0.3--0.4 mT], and in the Inhibited signal coupling was weaker still [A(33S)av. 0.1--0.2 mT]. Clearly, there must be substantial differences in the structures of the molybdenum centre in the different signal-giving species, with the sulphur atom perhaps in an equatorial type of ligand position in the Very Rapid species but in a more axial one in the other species. Structures are discussed in relation to the mechanism of action of the enzyme and the nature of the proton-accepting group that participates in turnover.
The interaction of copper(II) and glycyl-l-histidyl-l-lysine, a growth-modulating tripeptide from plasma
Authors: Lau S J , Sarkar B
Published: August 2015

The interaction between Cu(II) and the growth-modulating tripeptide glycyl-L-histidyl-L-lysine in the presence and absence of L-histidine was investigated by potentiometric titration and visible-absorption spectrophotometry at 25 degrees C in 0.15 M-NaCl. Analyses of the results in the pH range 3.5--10.6 indicated the presence of multiple species in solution in the binary system and extensive amounts of the ternary complexes in the ternary system. The species distribution and the stability constants, as well as the visible-absorption spectra of the species, were evaluated. The combined results were used to propose the structure of some of the complexes. The influence of the epsilon-amino group of the peptide in the enhancement of the stability constants was reflected prominently when compared with those complexes formed by either glycyl-L-histidine or glycyl-L-histidylglycine. The results obtained from the equilibrium-dialysis experiments showed that this tripeptide was able to compete with albumin for Cu(II) at pH 7.5 and 6 degrees C. At equimolar concentrations of albumin and the peptide, about 42% of the Cu(II) was bound to the peptide. At the physiologically relevant concentrations of Cu(II), albumin, L-histidine and this peptide, about 6% of the Cu(II) was associated with the low-molecular-weight components. This distribution could be due to the binary as well as the ternary complexes. The possible physiological role of these complexes in the transportation of Cu(II) from blood to tissues is discussed.
The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis
Authors: Gilbert H J , Jack G W
Published: August 2015

Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.
Evidence that lanthanum ions stimulate calcium inflow to isolated hepatocytes
Authors: Parker J C , Barritt G J
Published: August 2015

LaCl3 stimulated the initial rate of 45Ca2+ exchange measured under steady-state conditions in isolated liver cells. Cu2+ greater than La3+ = Fe3+ greater than Fe2+ = Zn2+ greater Ni2+ greater than Mn2+ also stimulated 45Ca2+ exchange. Compartmental analysis of 45Ca2+-exchange curves obtained in the presence or absence of La3+, and in the presence or absence of adrenaline, showed that the predominant effect of La3+ is to stimulate the inflow of Ca2+ to the cell from the medium. No evidence for an inhibition of Ca2+ outflow from the cell was obtained. In the presence of La3+, adrenaline caused no further stimulation of Ca2+ inflow to the cell. In the absence of adrenaline, La3+ increased the uptake of Ca2+ (measured by atomic-absorption spectroscopy) by isolated hepatocytes incubated at 1 degree C. The proposal that La3+ stimulates Ca2+ inflow to the liver cell by inducing a conformational change in the Ca2+-inflow transporter of the plasma membrane is briefly discussed.
Combined radiolabel-binding and immunocytochemical evaluation of receptor–ligand interactions. Studies of transferrin receptors on activated lymphocytes
Authors: Galbraith Gillian M. P. , Galbraith Robert M.
Published: August 2015

A protocol that involved both immunohistological and radiolabel-binding procedures was devised for the study of transferrin–receptor interactions. This composite approach yielded considerably more information than did either technique used alone, and also provided a simple means for exclusion of several common potential sources of error.
Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells
Authors: Dahlig-Harley E , Eilam Y , Paterson A R P , Cass C E
Published: August 2015

Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.
Effect of diethylstilboestrol on phosphatidylcholine biosynthesis and choline metabolism in the liver of roosters
Authors: Vigo C , Vance D E
Published: August 2015

It has been known for 40 years that oestrogens stimulate phospholipid metabolism in roosters. We have investigated in vivo the mechanism for this effect. Young roosters were injected daily with 1 mg of diethylstilboestrol for 1--3 days. At 4 h after the last injection, 30 microCi of [Me-3H]choline was injected into the portal vein. At periods up to 3 min the livers were freeze-clamped and choline and its metabolites were extracted and resolved by t.l.c. Hormone treatment in the first 2 days resulted in a 2-fold increase in phosphorylation of [Me-3H]choline and a decrease in the oxidation of [Me-3H]choline to [3H]betaine. The concentrations of phosphocholine in liver were increased 2-fold during the first 2 days concomitant with a 2-fold increase in the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, many of the above effects were reversed and the rate of phosphatidylcholine biosynthesis decreased to approx. 60% of the control value. The results suggest that the initial hormone treatments activate choline kinase within 4 h and, thereby, divert choline form oxidation to betaine. The resulting increased phosphocholine concentrations cause an increase in the activity of CTP:phosphocholine cytidylyltransferase, which results in a doubling of the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, the biosynthesis of phosphatidylcholine is decreased, most likely by an effect on the cytidylyltransferase reaction.
Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg
Authors: Yarlett N , Hann A C , Lloyd D , Williams A
Published: August 2015

This paper reports for the first time the presence in the anaerobic rumen ciliate Dasytricha ruminantium (Schuberg) of microbody-like organelles, about 0.5 micrometer diameter, with a granular matrix and an equilibrium density of approx. 1.18 g/ml. These organelles can be isolated in a fraction sedimented at 10(5) g-min that contains 67% of the total pyruvate synthase (EC 1.2.7.1), 66% of the hydrogenase (EC 1.18.3.1) and 20% of the lactate dehydrogenase (EC 1.1.1.27). Thus in several respects this fraction is enzymically similar to those containing hydrogenosomes in some other parasitic anaerobic protozoa (the trichomonads). However, in contrast with the hydrogenosomes of trichomonads, the oxygen-tolerant enzyme malate dehydrogenase (decarboxylating) (EC 1.1.1.40) is not particulate, but occurs only in the cytosol. These results enable the proposal of a scheme for the pathway of product formation (acetate, lactate, CO2 and H2) from carbohydrates.
Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7)
Authors: Geier A , Haimsohn M , Malik Z , Lunenfeld B
Published: August 2015

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.
Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli
Authors: Daruwalla K R , Paxton A T , Henderson P J
Published: August 2015

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.
Epithelial sodium channel (ENaC) is multi-ubiquitinated at the cell surface
Authors: Wiemuth Dominik , Ke Ying , Rohlfs Meino , Mc Donald Fiona J.
Published: August 2015

The human ENaC (epithelial sodium channel), a complex of three subunits, provides the rate-limiting step for sodium uptake in the distal nephron, and therefore plays a key role in salt homoeostasis and in regulating blood pressure. The number of active sodium channel complexes present at the plasma membrane appears to be tightly controlled. In Liddle's syndrome, a form of hypertension caused by an increase in the number of active sodium channels at the cell membrane, the βENaC or γENaC subunit gene contains a mutation that disrupts the binding site for the Nedd4 (neuronal precursor cell expressed developmentally down-regulated gene 4) family of ubiquitin-protein ligases. Therefore ubiquitination of channel subunits may be involved in altering cell surface ENaC. Here, we provide evidence that the ENaC subunits located at the cell surface are modified with multiple mono-ubiquitins (multi-ubiquitination) and that Nedd4-2 modulates this ubiquitination. We confirm that ENaC is associated with the μ2 subunit of the AP-2 (adaptor protein 2) clathrin adaptor. Since mono- or multi-ubiquitination of other membrane proteins is a signal for their internalization by clathrin-mediated endocytosis and subsequent trafficking, our results support a model whereby ubiquitin and clathrin adaptor binding sites act in concert to remove ENaC from the cell surface.
STAT5 isoforms: controversies and clarifications
Authors: Ramos Haydeé L. , O'Shea John J. , Watford Wendy T.
Published: August 2015

STAT (signal transducer and activator of transcription) family transcription factors are critical regulators of the development and differentiation of many cell types. STAT isoforms are generated by alternative splicing, but have also been suggested to be generated post-transcriptionally. In this issue of the Biochemical Journal, Schuster and colleagues have identified cathepsin G as the protease that cleaves full-length STAT5 (STAT5α) to generate a C-terminally truncated form in immature myeloid cells. However, the authors argue that this proteolytically generated isoform does not occur naturally in vivo; rather, it is artificially generated by cathepsin G during the preparation of cell extracts. This new evidence calls into question the physiological significance of this putative isoform and forces the general re-examination of proteolytically generated STAT isoforms.
Kinetic mechanism of mitochondrial adenosine triphosphatase. Inhibition by azide and activation by sulphite
Authors: Vasilyeva E A , Minkov I B , Fitin A F , Vinogradov A D
Published: August 2015

1. The initial rapid phase of ATP hydrolysis by bovine heart submitochondrial particles or by soluble F1-ATPase is insensitive to anion activation (sulphite) or inhibition (azide). 2. The second slow phase of ATP hydrolysis is hyperbolically inhibited by azide (Ki approximately 10(-5) M); the inosine triphosphatase activity of submitochondrial particles or F1-ATPase is insensitive to azide or sulphite. 3. The rate of interconversion between rapid azide-insensitive and slow azide-sensitive phases of ATP hydrolysis does not depend on azide concentration, but strongly depends on ATP concentration. 4. Sulphite prevents the interconversion of the rapid initial phase of the reaction into the slower second phase, and also prevents and slowly reverses the inhibition by azide. 5. The presence of sulphite in the mixture when ADP reacts with ATPase of submitochondrial particles changes the pattern of the following activation process. 6. Azide blocks the activation of ATP-inhibited ATPase of submitochondrial particles by phosphoenolpyruvate and pyruvate kinase. 7. The results obtained suggest that the inhibiting effect of azide on mitochondrial ATPase is due to stabilization of inactive E*.ADP complex formed during ATP hydrolysis; the activation of ATPase by sulphite is also realized through the equilibrium between intermediate active E.ADP complex and inactive E*.ADP complex.
The binding of rat uterine cytosol oestrogen receptors to oligodeoxythymidylate-cellulose. Its relationship to a stable form of receptor complex with separate ligand- and oligonucleotide-binding sites
Authors: Myatt L , Elder M G , Neethling C , Lim L
Published: August 2015

The interaction of rat uterine cytosol oestrogen-receptor complexes with the synthetic acceptor oligo(dT)-cellulose was studied. Differences in the stability of receptor complexes and their ability to bind to oligo(dT)-cellulose on storage at 4 degrees C or when exposed to increased temperatures indicated heterogeneity of steroid- and oligonucleotide-binding sites. Dilution, dialysis and (NH4)2SO4 precipitation increased the interaction of receptor complexes with oligo(dT)-cellulose (a step termed activation). This increase may be the result of the removal of low-molecular-weight cytosol components which inhibit receptor activation, dimerization to the 5 S form, which binds to oligo(dT)-cellulose, or interaction of 5 S receptor with the oligonucleotide. Cytosol oestradiol-receptor complexes exhibited biphasic dissociation kinetics. All these manipulations resulted in an increase in the proportion of the slow-dissociating component equivalent to the increase in receptor binding to oligo(dT)-cellulose. In contrast, addition of 10mM-sodium molybdate to cytosol decreased both oligo(dT)-cellulose binding and the proportion of receptor with slow dissociation kinetics. The inclusion of proteinase inhibitors did not affect interactions of receptor with oligo(dT)-cellulose nor the dissociation kinetics. These results suggest that oligo(dT)-cellulose binding may serve to quantify the proportion of cytosol receptor in an active form capable of nuclear interaction and to help to ascertain whether a receptor system is fully functional. This binding procedure could prove useful in the evaluation of oestrogen responsivity under normal and pathological conditions.
Teichoic acid and lipid metabolism during sporulation of Bacillus megaterium KM
Authors: Johnstone K , Simion F A , Ellar D J
Published: August 2015

The biochemistry of teichoic acid and lipid metabolism has been studied during sporulation of Bacillus megaterium KM. Measurements of cell-wall and membrane teichoic acid have shown that net synthesis of these polymers ceases at the onset of sporulation. Pulse-labelling studies show that the period of asymmetric septation and forespore engulfment is marked by an initiation of turnover of membrane teichoic acid but not of wall teichoic acid. This is reflected in the presence of inner-membrane teichoic acid and the virtual absence of wall teichoic acid in dormant spores. The total amount of lipid phosphorus in the sporulating cell increases by 70% as a result of asymmetric septation and subsequent engulfment of the forespore. The phosphorus requirement for this synthesis is derived from a pool formed during exponential growth, which is not exchangeable with extracellular Pi during sporulation. These results suggest that during sporulation a proportion of the glycerol 3-phosphate produced by preferential degradation of membrane teichoic acid formed during exponential growth is used for phospholipid synthesis during sporulation.
Studies of lactate dehydrogenase in the purified state and in intact erythrocytes
Authors: Simpson R J , Brindle K M , Brown F F , Campbell I D , Foxall D L
Published: August 2015

Lactate dehydrogenase in intact erythrocytes was studied by observing isotope exchange between lactate and pyruvate by p.m.r. The inhibition of the enzyme in intact cells by both oxalate and pyruvate was found to be similar to that of the purified enzyme. The activity of the enzyme in intact cells indicates that the free solution NAD+ + NADH concentration in erythrocytes is about 10 microM whereas the total extractable NAD+ + NADH is about 80 nmol/ml of cell water.
Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein
Authors: Dix Clive J. , Schumacher Matthias , Cooke Brian A.
Published: August 2015

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED50 (dose that produces a response that is 50% of the maximum response) 60±5.7ng/ml and 8±1.8ng/ml (mean±s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/106 cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific 125I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4°C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5′-[β,γ-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50–60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.
Use of radioactive glucosamine in the perfused rat liver to prepare α1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues
Authors: Aronson N N
Published: August 2015

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.
A method for determining kinetic parameters at high enzyme concentrations
Authors: Halfman C J , Marcus F
Published: August 2015

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.