bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats
Authors: Sömjen D , Sömjen G J , Weisman Y , Binderman I
Published: August 2015

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.
The induction of synthesis of L-type pyruvate kinase in cultured rat hepatocytes
Authors: Poole G P , Postle A D , Bloxham D P
Published: August 2015

Hepatocytes were isolated from preweaned neonatal and adult rats and maintained in primary monolayer culture. Cells from preweaned newborns possessed no L-type pyruvate kinase, nor did they synthesize the enzyme. Incubation for 48-72 h in culture medium supplemented with 2 mM-fructose and 0.1 microM-insulin induced the synthesis of L-type pyruvate kinase, as judged by increased enzyme activity and the increased incorporation of [3H]leucine into immunoprecipitable L-type pyruvate kinase. Hepatocytes isolated from 48 h-starved adult rats incorporated less [3H]leucine into L-type pyruvate kinase than did cells isolated from high-carbohydrate-diet-fed rats. The rate of enzyme synthesis by cells from 48 h-starved rats was increased by the inclusion of fructose and insulin in the incubation medium, after a lag phase of 24-48 h. After 4 days in culture in the presence of fructose and insulin, hepatocytes from 48 h-starved rats synthesized L-type pyruvate kinase at similar rates to hepatocytes isolated from high-carbohydrate-diet-fed rats.
Kinetic evidence for a common mechanism of capping on lymphocytes
Authors: Corps Anthony N. , Metcalfe James C. , Pozzan Tullio
Published: August 2015

1. Differences in the rates at which ligands cap various receptors on the same cells, and their sensitivity to various drugs, have been interpreted as evidence that there are distinct mechanisms for ‘fast’ and ‘slow’ cap formation. We have examined the factors which determine the rate of cap formation of three receptors on mouse splenic lymphocytes or thymocytes, and compared the effects of cytochalasin B or colchicine under conditions where the different receptors cap at similar rates. 2. When surface immunoglobulin, concanavalin A receptors, or θ antigen are induced to cap at their maximal rates by appropriate concentrations of one or more cross-linking ligands, the half-time for maximal capping of each receptor population is between 1.5 and 3.0min at 37°C. Slower rates of cap formation are obtained by using non-optimal concentrations of the cross-linking ligands. 3. When the three receptors were induced to cap at similar rates (either maximal or slower), 10μm-cytochalasin B caused a similar decrease in the rate of cap formation for each receptor, without affecting the eventual extent of capping. At comparable capping rates on control cells, colchicine (10μm) increased the rate of cap formation for surface immunoglobulin and concanavalin A receptors to a similar extent, without affecting the eventual extent of cap formation. In contrast, colchicine had no detectable effect on the capping of θ antigen. 4. From these results, we conclude that there are no intrinsic differences in the rates at which different receptors can be induced to cap that can be used to diagnose differences in their mechanisms of cap formation. The observation that ligand concentration and the drugs acting on the cytoskeleton generally affect the rate but not the extent of cap formation accounts for the wide variation in reported effects of the drugs on cap formation measured at fixed times. The receptor-specific effect of colchicine on surface immunoglobulin and concanavalin A receptors, but not θ antigen, is not readily compatible with models of cap formation which depend on lipid or membrane flow.
Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in rat liver and Morris hepatomas 5123C, 9618A and 5123t.c.
Authors: Gregg R G , Sabine J R , Wilce P A
Published: August 2015

Characteristics of 3-hydroxy-3-methylglutaryl-CoA reductase from normal liver, Morris hepatomas 5123C, 5123t.c. and 9618A, and host liver were studied. Animals were fed on control and 5%-cholesterol diets. Microsomal membranes from all tissues were found to accumulate cholesterol after 3 days on the 5%-cholesterol diet. The enzyme of the tumours showed no feedback inhibition by dietary cholesterol, and that of host liver gave a variable response, whereas that of control liver was constantly inhibited by 90% or more. Arrhenius-plot analysis was conducted on the microsomal enzyme isolated from the various tissues. Control animals showed that the phase transition present at 27 degrees C was removed when animals were fed on 5%-cholesterol diet for 12 h. The hepatomas failed to show this change even after 3 days of 5%-cholesterol diet and a significant increase in microsomal cholesterol. This failure to remove the break in Arrhenius plots also occurred in host liver, even though enzyme inhibition occurred. The reason why hepatomas fail to regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in response to dietary cholesterol may be a decreased membrane-enzyme interaction.
Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat
Authors: Albano E , Lott K A K , Slater T F , Stier A , Symons M C R , Tomasi A
Published: August 2015

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide (‘phenyl t-butyl nitrone’) (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.
Effect of 5‘-deoxy-5’-isobutylthioadenosine on putrescine uptake and polyamine biosynthesis by chick embryo fibroblasts
Authors: Lawrence F , Bachrach U , Robert-Géro M
Published: August 2015

The effect of 5‘-deoxy-5’-S-isobutylthioadenosine (SIBA) on polyamine biosynthesis has been studied by using cultured chick embryo fibroblasts. It has been shown that the drug inhibits the uptake of [14C]putrescine and its conversion into labelled spermidine or spermine. The inhibitory effect is reversed by removing the inhibitor after exposing the cells to the drug for 24 h. SIBA also caused a significant decrease in cellular spermine levels and an accumulation of putrescine. These changes are reversed by removing the inhibitor. SIBA had the same effect on chick embryo fibroblasts transformed by Rous sarcoma virus; a decrease in cellular spermine levels in SIBA-treated cells was observed. In all the experiments SIBA caused a reduction in the spermine/putrescine and spermidine/putrescine ratios. It is suggested that SIBA is not only an inhibitor of transmethylation but also interferes with polyamine biosynthesis, probably by blocking aminopropyltransferase.
Effect of inhibition of fatty acid oxidation on neonatal liver carnitine content
Authors: Frost Susan C. , Wells Michael A.
Published: August 2015

The hypoglycaemic agent 2-tetradecylglycidic acid (compound McN-3802) caused an increase in total liver carnitine content, this being due primarily to an increase in the free carnitine pool. In the neonatal animal, this may represent a mechanism to overcome the inhibitory effect of fatty acid oxidation by the drug.
Transitions in human atrial and ventricular myosin light-chain isoenzymes in response to cardiac-pressure-overload-induced hypertrophy
Authors: Cummins P
Published: August 2015

1. The light-chain subunits of human atrial and ventricular cardiac muscle were examined by two-dimensional polyacrylamide-gel electrophoresis and limited proteolytic digestion. The light-chain patterns in the normal right and left atria were identical. 2. Myosin preparations isolated from right or left atria that had been subjected to cardiac-pressure-overload-induced hypertrophy also contained ventricular light-chain subunits. These were identified by peptide mapping in sodium dodecyl sulphate. 3. Ventricular light chain-2 was the major species in hypertrophied atria, although light chain-1 subsequently appeared in severe pressure-overload-hypertrophied cases. Evidence is presented for the existence of more than one form of ventricular light chain-2. 4. The transition from atrial to ventricular myosin light chains correlated with the degree of pressure-overload hypertrophy in 83 examples of surgically excised atria. 5. The adult atrial light chain-1 was shown to be homologous to the human foetal ventricular light chain-1 [Price, Littler & Cummins (1980) Biochem. J. 191, 571-580] by peptide mapping. 6. A scheme of atrial/ventricular myosin light-chain isoenzyme transitions is discussed in relation to changing contractile properties in cardiac muscle, together with implications for the role of light-chain subunits.
Does superoxide anion participate in 2-oxoglutarate-dependent hydroxylation?
Authors: Holme Elisabeth , Lindstedt Göran , Lindstedt Sven , Nordin Ingalill
Published: August 2015

The possible role of superoxide anion in 2-oxoglutarate-coupled dioxygenase reactions has been investigated. γ-Butyrobetaine hydroxylase (EC 1.14.11.1) was inhibited by human erythrocyte superoxide dismutase (EC 1.15.1.1), probably due to release of Cu2+ or Zn2+, as the inhibition was more pronounced after heat-inactivation of the dismutase and as Cu2+ was a potent inhibitor. Bovine superoxide dismutase and the Mn2+-containing superoxide dismutase from Escherichia coli were not inhibitory. Superoxide anion generated from xanthine/xanthine oxidase was not stimulatory and could not replace ascorbate. Thymine 7-hydroxylase (EC 1.14.11.6) and thymidine 2′-hydroxylase (EC 1.14.11.3) were not inhibited by erythrocyte superoxide dismutase or stimulated by superoxide anion. γ-Butyrobetaine hydroxylase was inhibited by a number of low-molecular-weight compounds, such as tetranitromethane, Nitro Blue Tetrazolium, adrenaline and Tiron, which may act as scavengers of superoxide anion. Involvement of this radical in other oxygenase reactions has been inferred from the findings that they were inhibitory for the respective enzymes. Several of these compounds also inhibited γ-butyrobetaine hydroxylase. It could be concluded from these experiments, however, that mechanisms other than disposal of superoxide anion might equally well be operative, such as hydrophobic interaction with the enzyme protein and interaction with compounds required for full enzymic activity, e.g. iron and ascorbate. The results appear to rule out a requirement for superoxide anion generated in free solution, and have not yielded evidence for participation of enzyme-bound superoxide anion in 2-oxoglutarate-dependent hydroxylations.
Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies
Authors: Emanuel E J , Brampton A D , Burton D R , Dwek R A
Published: August 2015

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q-immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15-0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9-12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338-344].
Identification of two major proteins of bovine pancreatic stones as immunoreactive forms of trypsinogens
Authors: De Caro A , Multigner L , Vérine H
Published: August 2015

Two major proteins have been identified in sodium citrate extracts of bovine pancreatic stones from 15 glands with lithiasis. They were found to have a molecular weight of about 24 000 and were further characterized by a variety of methods, including polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, isoelectric focusing, two-dimensional electrophoresis, immunodiffusion, immunoelectrophoresis and determination of N-terminal residues. These two immunologically and electrophoretically different proteins were definitely shown to be immunoreactive forms of anionic and cationic trypsinogens, which are normal components of pancreatic juice. However, in contrast with both secretory trypsinogens, the stone proteins displayed an important charge heterogeneity under isoelectric-focusing conditions. A possible role for both secretory trypsinogens in pancreatic lithogenesis is suggested by the reproducibility of the data. Finally, two minor proteins with a lower molecular weight (about 11 000-13 000) have also been found to be present in all extracts, but have not yet been identified.
Carbonic anhydrase C in white-skeletal-muscle tissue
Authors: Siffert W , Gros G
Published: August 2015

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.
Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte
Authors: Wakeyama H , Takeshige K , Takayanagi R , Minakami S
Published: August 2015

A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at −70 degrees C.
Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes
Authors: García Gil Merche , Alonso Fernando , Alvarez Chiva Vicente , Sánchez Crespo Mariano , Mato José M.
Published: August 2015

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun.101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-14C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.
Role of alcohol dehydrogenase activity and the acetaldehyde in ethanol- induced ethane and pentane production by isolated perfused rat liver
Authors: Müller A , Sies H
Published: August 2015

The volatile hydrocarbons ethane and n-pentane are produced at increased rates by isolated perfused rat liver during the metabolism of acutely ethanol. The effect is half-maximal at 0.5 mM-ethanol, and its is not observed when inhibitors of alcohol dehydrogenase such as 4-methyl- or 4-propyl-pyrazole are also present. Propanol, another substrate for the dehydrogenase, is also active. Increased alkane production can be initiated by adding acetaldehyde in the presence of 4-methyl- or 4-propyl-pyrazole. An antioxidant, cyanidanol, suppresses the ethanol-induced alkane production. The data obtained with the isolated organ demonstrate that products known to arise from the peroxidation of polyunsaturated fatty acids are formed in the presence of ethanol and that the activity of alcohol dehydrogenase is required for the generation of the active radical species. The mere presence of ethanol, e.g. at binding sites of special form(s) of cytochrome P-450, it not sufficient to elicit an increased production of volatile hydrocarbons by rat liver.
Enhancement of rat brain catecholamine synthesis by administration of small doses of tyrosine and evidence for substrate inhibition of tyrosine hydroxylase activity by large doses of the amino acid
Authors: Badawy Abdulla A.-B. , Williams David L.
Published: August 2015

Rat brain catecholamine synthesis is enhanced by small doses of tyrosine, but not by doses of 50mg/kg body wt. and above. It is suggested that these latter doses overcome the above enhancement by causing a substrate inhibition of tyrosine hydroxylase activity.
Effects of starvation on the maximal activities of some glycolytic and citric acid-cycle enzymes and glutaminase in mucosa of the small intestine of the rat
Authors: Budohoski Leszek , Challis R. A. John , Newsholme Eric A.
Published: August 2015

Starvation decreases activities of some glycolytic and citric acid-cycle enzymes, and increases those of glucose 6-phosphatase and fructose bisphosphatase, whereas that of glutaminase is unchanged. These findings may be of significance for the control of glucose metabolism in the absorptive cells of the intestine.
Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry
Authors: Gay R J , Amos H
Published: August 2015

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.
Dynamics of carbon monoxide recombination to fully reduced cytochrome c oxidase in plant mitochondria after low-temperature flash photolysis
Authors: Denis M , Richaud P
Published: August 2015

Rebinding of CO to reduced cytochrome c oxidase in plant mitochondria has been monitored optically at 590-630 nm after flash photolysis at low temperature from 160 to 200 K. (1) Under 100%-CO saturation, CO rebinding exhibits a four-step mechanism. The thermodynamic parameters of the first phase have been determined; its activation energy, Ea1, is 38.9 kJ.mol-1 and its enthalpy, delta H+/-1, and entropy, delta S+/-1, of activation are respectively 37.5 kJ.mol-1 and −75.8J.mol-1.K-1. (2) When the CO concentration is decreased to 0.2%, rebinding still occurs according to a four-step mechanism. The rate constant of the first phase is CO-concentration-independent. Under non-saturating conditions there is only one CO molecule per occupied site. The rebinding mechanism does not require additional CO molecules to be present in the haem pocket. (3) Dual-wavelength scanning experiments failed to detect optical forms correlated with the resolved phases. (4) Results are discussed with respect to previous work related to CO rebinding to mammalian cytochrome c oxidase and myoglobin.
Ethylene production from α-oxo-γ-methylthiobutyric acid is a sensitive measure of ligninolytic activity by Phanerochaete chrysosporium
Authors: Kelley R L , Reddy C A
Published: August 2015

Production of hydroxyl radical in ligninolytic cultures was determined by measuring the alpha-oxo-gamma-methylthiobutyric acid-dependent production of ethylene gas. The results showed that the pattern of ethylene production was very similar to that of ligninolytic activity [[14C]lignin leads to 14CO2). Furthermore, nutritional parameters, which are known to affect ligninolytic activity, affected OH.-radical-dependent ethylene production in a similar fashion. The results indicate that assay for ethylene production from alpha-oxo-gamma-methylthiobutyric acid is a simple and sensitive measure of ligninolytic activity by Phanerochaete chrysosporium.
Methyl group transfer from exogenous S-adenosylmethionine on to plasma-membrane phospholipids without cellular uptake in isolated hepatocytes
Authors: Van Phi L , Söling H D
Published: August 2015

At external concentration of 50 microM, L-methionine was rapidly taken up by hepatocytes, whereas almost no S-adenosylmethionine (SAM) was removed from the incubation medium. SAM did not enter the intracellular water space but equilibrated with a very small pool, which was most likely to be situated on the external side of the plasma membrane. Methyl groups from external L-methionine, but not from external SAM, were incorporated into total and nuclear RNA. A significant incorporation of methyl groups into phospholipids occurred not only with methionine but also with SAM. After subfractionation of hepatocytes it became evident that methyl groups from SAM were mainly incorporated into plasma-membrane phospholipids, and that phospholipid methylation in other cellular compartments resulted from contamination with plasma membrane. The pattern of methylation of the various phospholipid species with SAM as precursor was different from that obtained with L-methionine. In contrast with external L-methionine, external SAM did not enter the intracellular SAM pool. According to these results a transport system for SAM does not exist in rat hepatocytes, although methyl groups from external SAM can be incorporated into plasma-membrane phospholipids from the outside.
A comparative study of the localization of wheat-germ agglutinin and its potential receptors in wheat grains
Authors: Miller R C , Bowles D J
Published: August 2015

Wheat-germ agglutinin is located only in the embryo of a dry wheat seed and not in the endosperm tissue. This distribution remains unaltered for up to 96 h of germination and growth. The lectin is found not only in a freely soluble form but also in reversible association with particulate subcellular components. There appear to be no poly-peptides that can be solubilized with sonication and aqueous buffers from the embryo tissue that can interact with the agglutinin. This suggests that in vivo the lectin remains uncomplexed to endogenous glycoconjugates or is only able to bind to glycosylated integral membrane polypeptides. Alternatively the potential endogenous receptor(s) to wheat-germ agglutinin may not contain a polypeptide. Although the lectin is not present in the endosperm, seven polypeptides able to interact in a reversible way with wheat-germ agglutinin could be purified from that tissue.
Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes
Authors: Imam A , Laurence D J , Neville A M
Published: August 2015

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed ‘epithelial-membrane glycoprotein’ (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.
Multiple binding of thallium and rubidium to potassium-activated yeast aldehyde dehydrogenase. Influences on tertiary structure, stability and catalytic activity
Authors: Bostian K A , Betts G F , Man W K , Hughes M N
Published: August 2015

Univalent cation activators of aldehyde dehydrogenase have dual effects, both interpreted as cation-induced or -stabilized conformation changes. These two processes are differentiated by the time scales of their associated changes in activity. Using Tl+ as an activator, under certain conditions, the slower change in activity saturates at a Tl+ concentration which is only 0.1 Ks for the faster change. This, together with evidence for cation-induced rather than cation-stabilized conformation changes, is used to propose separate binding sites for cations responsible for the two activation processes. Equilibrium dialysis indicates 4 binding sites per active site for Rb+ or 6 sites for Tl+. At least one of the additional sites for Tl+ is an inhibitory site which has been differentiated from the activator sites on the basis of steady-state and pre-steady-state kinetic data.
A comparison of the catalytic activities of human plasma kallikreins I and II
Authors: Levison P R , Tomalin G
Published: August 2015

Subsites in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] were identified in human plasma kallikrein II (EC 3.4.21.8). Kinetic constants (kcat, Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. With these substrates it was found that deacylation of the enzyme was rate-limiting. It was possible to infer that L-proline at residue P3 interacted with subsite S3 of the enzyme and L-phenylalanine at residue P2 interacts hydrophobically with subsite S2 in addition to hydrogen-bonded interactions with this subsite region. By comparison with the results of a similar study with human plasma kallikrein I, it is observed that although broadly similar subsite interactions occur between the two enzyme forms, the rate of deacylation of kallikrein II is approx. 35% of that observed for kallikrein I, and the latter form is up to ten times more active (in terms of kcat./Km) than kallikrein II.
Amino acid sequences of two novel long-chain neurotoxins from the venom of the sea snake Laticauda colubrina
Authors: Kim H S , Tamiya N
Published: August 2015

From the venom of a population of the sea snake Laticauda colubrina from the Solomon Islands, a neurotoxic component, Laticauda colubrina a (toxin Lc a), was isolated in 16.6% (A280) yield. Similarly, from the venom of a population of L. colubrina from the Philippines, a neurotoxic component, Laticauda colubrina b (toxin Lc b), was obtained in 10.0% (A280) yield. The LD50 values of these toxins were 0.12 microgram/g body wt. on intramuscular injection in mice. Toxins Lc a and Lc b were each composed of molecules containing 69 amino acid residues with eight half-cystine residues. The complete amino acid sequences of these two toxins were elucidated. Toxins Lc a and Lc b are different from each other at five positions of their sequences, namely at positions 31 (Phe/Ser), 32 (Leu/Ile), 33 (Lys/Arg), 50 (Pro/Arg) and 53 (Asp/His) (residues in parentheses give the residues in toxins Lc a and Lc b respectively). Toxins Lc a and Lc b have a novel structure in that they have only four disulphide bridges, although the whole amino acid sequences are homologous to those of other known long-chain neurotoxins. It is remarkable that toxins Lc a and Lc b are not coexistent at the detection error of 6% of the other toxin. Populations of Laticauda colubrina from the Solomon Islands and from the Philippines have either toxin Lc a or toxin Lc b and not both of them.
Appendix: Synthesis of N-acylglutathione derivatives by dimethylaminopyridine catalysis
Authors: D'Silva Claudius , Al-Timari Amina , Douglas Kenneth T.
Published: August 2015

A general procedure, involving 4-dimethylaminopyridine-catalysed acylation of the amino groups in the appropriate S-blocked glutathione, is reported for the preparation of N-blocked derivatives of glutathiones.
Steady-state kinetic studies of the inhibitory action of Zn2+ on ribonuclease T1 catalysis
Authors: Itaya M , Inoue Y
Published: August 2015

The kinetic mechanism of specific inhibition by Zn2+ of ribonuclease T1 catalysis was studied by steady-state kinetic analysis of transphosphorylation of dinucleotides, GpCp(3′), GpUp(2′) and GpUp(3′), and dinucleoside monophosphates, GpC and GpU. The inhibition was not simply competitive, non-competitive or uncompetitive, but the kinetic data were compatible with a mechanism of ‘fully mixed inhibition’ in which a fully non-competitive action was associated with a partially competitive action. Apparent equilibrium quotients involved in this model of inhibition were determined for the dinucleotide substrates, and we found that binding of either of Zn2+ and substrate was facilitated when the other was bound. The location of Zn2+ was suggested to be near His-40 and/or His-92 of the ribonuclease T1 molecule.
Evidence that enteroglucagon (II) is identical with the C-terminal sequence (residues 33-69) of glicentin
Authors: Holst J J
Published: August 2015

Enteroglucagon (II) was isolated from extracts of pig ileum mucosa by repeated gel filtrations, and its immunochemical and chromatographic characteristics were compared with those of a synthetic peptide corresponding to the 33-69 sequence of pig glicentin, before and after digestion with trypsin or trypsin followed by carboxypeptidase B, by using five region-specific assays covering most of the glicentin sequence. Enteroglucagon (II) and the synthetic peptide behave identically under three different conditions of chromatography as determined with all five assays (including a highly specific radioreceptor assay), and gave rise to similar fragments after enzyme digestion. It was therefore concluded that enteroglucagon (II) and the 33-69 sequence of glicentin are most probably identical.
The determination by radiochemical assay of argininosuccinase produced in an Escherichia coli system in vitro
Authors: Zidwick M J , Rogers P
Published: August 2015

A highly sensitive radiochemical assay used to measure the synthesis and regulation of the product of the argH gene, argininosuccinase, in an Escherichia coli system in vitro is described. With L-[guanidino-14C]argininosuccinic acid as a substrate, and in the presence of excess arginase and urease, 14CO2 is collected in a simply designed micro-vessel. With this method less than 1 nmol of product can be measured in the presence of various concentrations of L-arginine.
Enzymes of mercapturic acid production in rat mammary gland
Authors: Puente J , Castellon E , Sapag-Hagar M
Published: August 2015

1. The enzymes glutathione S-transferase, gamma-glutamyl peptidyltransferase and dipeptidase, which participate in the detoxification pathway through mercapturic acid production, were measured in rat mammary gland during pregnancy and lactation. 2. Mammary-gland concentration of reduced glutathione showed, concomitantly with the enzyme activities, a significant increase during lactation. 3. The mammary-gland glutathione S-transferase exhibits characteristics quite similar to those described for the liver and kidney enzymes with respect to substrates, isoenzymes, molecular weight and probenecid and bilirubin inhibition. 4. In view of these similarities, mammary-gland glutathione S-transferase may play the same role as a cytoplasmic organic-anion receptor proposed for the hepatic enzyme. It may also represent a detoxification pathway for protecting the mammary tissue during the lactogenic cycle.
An enzyme-linked immunoassay for the collagen cross-link pyridinoline
Authors: Robins S P
Published: August 2015

An inhibition immunoassay method for the determination of pyridinoline was developed with the use of microtitre plates coated with a pyridinoline-gelatin conjugate and rabbit antisera directed against pyridinoline linked to bovine serum albumin. The sensitivity of the assay is about 2pmol of pyridinoline, and the presence of related pyridinium and lysine-derived compounds does not significantly interfere with the procedure. Its application to tissue and human urine samples is described.
Flow cytometric analysis of lectin binding to human peripheral blood lymphocytes
Authors: Blackledge G , Vose B , Morris A J , Crowther D , Gallagher J T
Published: August 2015

The binding of fluorescein-conjugated lentil lectin and concanavalin A to the surface membrane of human peripheral blood lymphocytes was studied by flow cytometry. The lymphocytes bound 3-fold more lentil lectin molecules compared with concanavalin A molecules and lentil lectin binding approached saturation at a much lower concentration than did that of concanavalin A. Lentil lectin identified two groups of lymphocytes: a low-binding T-cell fraction and a high-binding B-cell-enriched fraction. Concanavalin A did not discriminate between these populations in unseparated lymphocytes. Competition studies indicated that lentil lectin and concanavalin A were bound to different sites on the lymphocyte surface, although about 50% of lentil lectin sites were in close proximity to concanavalin A sites.
Transfer RNA and ribosomal RNA are synthesized from the same pyrimidine nucleotide pool
Authors: Birch B , Turnock G
Published: August 2015

We have tested the hypothesis [Wiegers, Kramer, Klapproth & Hilz (1976) Eur. J. Biochem. 64. 535-540] that the synthesis of rRNA in the nucleolus may have a partially independent supply of nucleoside triphosphates that is not completely suppressed when cells are supplied with exogenous nucleosides. For the slime mould, Physarum polycephalum, and Chinese hamster ovary cells in culture, the specific activities (3H/32P) of UMP and CMP in tRNA, which is synthesised in the nucleoplasm, and rRNA were compared after continuous labelling with [3H]uridine and [32P]phosphate. No differences were found, suggesting that transcription throughout the nucleus draws on a common supply of pyrimidine triphosphates that is uniformly labelled from exogenous [3H]uridine. As a control of the radioactive labelling schedule, a similar experiment was carried out with the prokaryote, Escherichia coli, and identical results were obtained.
Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes
Authors: Donner David B.
Published: August 2015

Native insulin inhibits the binding and degradation of 125I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of 125I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of 125I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of 125I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact.
Stimulating role of potassium ions and ouabain on glycogen synthesis in adipose tissue
Authors: Sobrino F , Ruiz G , Goberna R
Published: August 2015

1. Exposure of fat-pads to increasing concentrations of K+ in the presence of insulin stimulates the incorporation of labelled glucose into glycogen. In the absence of hormone, only a slight incorporation of glucose into glycogen and slight glucose oxidation were detectable. 2. Ouabain alone, up to 100 microM, had no effect on synthesis of glycogen. Ouabain reinforced the effect of insulin on the conversion of glucose into glycogen in a Na+ medium and in a equimolar Na+-K+ medium, but not in a K+ medium. In addition, ouabain modified the optimal K+/Na+ ratio for glycogen synthesis. 3. The proportion of glycogen synthase in the active form was increased in a K+ medium, and a faster rate of conversion of synthase b into a was observed under these conditions. No difference was detected in the rate of inactivation of phosphorylase in a K+ or a Na+ medium. 4. Even though these results, taken together, are consistent with the proposed role of phosphorylase a in the regulation of synthase activation, the molecular mechanism of action of K+ in adipose tissue in increasing synthesis of glycogen cannot be explained simply by a faster inactivation of phosphorylase a. It is concluded that some undetermined effector(s) or signal could itself be a primary determinant for the greater activation of synthase observed in a K+ medium.
The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of l-malate and d-malate by luminal-membrane vesicles
Authors: Kragh-Hansen Ulrich , Jørgensen Karl Evald , Sheikh M. Iqbal
Published: August 2015

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J.208, 359–368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na+ gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na+-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl−>SO42−>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na+/substrate coupling ratio 3:1) by an Na+-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na+ in the medium.
Studies in vitro on shuttle systems of mouse spermatozoa
Authors: Burgos C , Coronel C E , de Burgos N M G , Rovai L E , Blanco A
Published: August 2015

Observations on systems reconstituted in vitro with different starting substrates (2-hydroxy-acids, 2-oxo-acids or leucine) indicate that a branched-chain 2-hydroxy-acid/2-oxo-acid shuttle for the transfer of reducing equivalents from cytosol to mitochondria may be operational in mouse sperm. Evidence is presented suggesting that the 2-oxo-acids produced by intramitochondrial oxidation of 2-hydroxy-acids ingressed from the cytosol can recycle back into the external phase. Observations in vitro demonstrate that, in addition to the branched-chain 2-hydroxy-acid/2-oxo-acid shuttle, the malate/aspartate system is also active in mouse sperm. On the contrary, the lactate/pyruvate redox couple does not appear to function as part of a shuttle system in mouse sperm mitochondria. The glycerol 3-phosphate shuttle probably is not functionally significant in mouse spermatozoa, since the activity of the ‘soluble’ glycerol 3-phosphate dehydrogenase is very low.