bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of rat adipocyte pyruvate dehydrogenase by palmitate. Protection against this effect by insulin in the presence of glucose
Authors: Sooranna S R , Saggerson E D
Published: August 2015

1. Adipocytes from rat epididymal fat-pads were incubated for 30 min with 5 mM-glucose and concentrations of lactate, pyruvate and amino acids typical of those found in rat plasma. 2. PDHa (active form of pyruvate dehydrogenase) activity was significantly increased after incubation of the cells with insulin (200 micro-i.u./ml), and decreased by incubation with palmitate (0.5–2 mM). 3. In the presence of insulin, palmitate did not decrease PDHa activity. 4. Dichloroacetate (1 mM) increased PDHa activity in the absence of palmitate to the same extent as did insulin. In the presence of dichloroacetate but the absence of insulin, palmitate decreased PDHa activity. In the presence of dichloroacetate and insulin, palmitate again did not decrease PDHa activity. 5. It is concluded that, in the presence of glucose, insulin has a strong protective action against inactivation of adipocyte PDHa by fatty acids.
Metabolism of palmitate in perfused rat liver. Effect of low and high ethanol concentrations at various concentrations of palmitate in the perfusion medium
Authors: Kondrup Jens , Lundquist Frank , Damgaard Stig E.
Published: August 2015

1. The effect of ethanol on the metabolism of [1-14C]palmitate in rat liver was investigated in a single-pass perfusion system at concentrations of 10mm- or 80mm-ethanol and 0.2mm- or 1mm-palmitate. 2. After the perfusion the hepatic lipid was isolated in subcellular fractions. The two major fractions contained triacylglycerol from cytoplasmic lipid droplets and from endoplasmic reticulum plus Golgi apparatus respectively. 3. In experiments with 0.2mm-palmitate perfusion with 10mm- or 80mm-ethanol did not measurably increase the esterification, and the oxidation was markedly decreased and the fatty acid uptake was not affected. 4. Perfusion with ethanol, at 1mm-palmitate, increased the fatty acid uptake, increased esterification and decreased oxidation. The effects of 10mm- and 80mm-ethanol were similar. The incorporation of [1-14C]palmitate into triacylglycerol in cytoplasmic lipid droplets was not affected statistically significantly by ethanol. Ethanol increased the incorporation of [1-14C]palmitate into di- and tri-acylglycerol in the membranous fraction. Estimated chemically, the contents of di- and tri-acylglycerol were only slightly affected by ethanol. These results suggest that the effect of ethanol was to increase the turnover of fatty acids in triacylglycerol rather than to increase its accumulation. 5. The results indicate that an increased concentration of fatty acids is more important for the formation of acute fatty liver in fed rats than are the direct effects of ethanol on hepatic fatty acid metabolism.
Translational activity of messenger ribonucleic acid isolated from unstimulated and phytohaemagglutinin-activated lymphocytes
Authors: Wallace D M , Jagus R , Benzie C R , Kay J E
Published: August 2015

Purified cytoplasmic poly(A)+ RNA isolated from unstimulated pig lymphocytes has the same ability to direct translation in a range of cell-free systems as the corresponding mRNA from 20h phytohaemagglutinin-activated lymphocytes. Additional methylation of the mRNA is not required for maximum protein synthesis in the wheat-germ cell-free system. Misleading results are obtained if the mRNA preparations used are not adequately purified, and a method suitable for routine assessment of the degree of purification achieved is described. Cell-free protein-synthesizing systems from unstimulated lymphocytes translate added lymphocyte mRNA with lower efficiency than do comparable systems from phytohaemagglutinin-activated lymphocytes, whatever the source of the mRNA used.
The effects of triton X-100 on the transfer of mannose, glucose and N-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver
Authors: Coolbear T , Mookerjea S , Hemming F W
Published: August 2015

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.
Effects of fructose concentration on carbohydrate metabolism, heat production and substrate cycling in isolated rat hepatocytes
Authors: Clark Dallas G. , Filsell Owen H. , Topping David L.
Published: August 2015

1. Hepatocytes from starved rats were incubated with 5mm-glucose, labelled uniformly with 14C and specifically with 3H at positions 1, 2, 3 or 6, and with fructose at concentrations of 2.5, 7.5 or 25mm. 2. In the absence of other substrates only 1% of the radioactivity initially present in [U-14C]glucose appeared in the metabolic products, CO2, lactate, pyruvate, amino acids and glycogen. 3. Fructose at 2.5mm caused a 30% increase in the glucose concentration and a 4-fold increase in the apparent oxidation of [U-14C]-glucose. 4. The formation of 3H2O from [1-3H]-, [2-3H]-, [3-3H]- or [6-3H]-glucose was 2.4, 4.3, 2.15 or 1.6% respectively in the control incubations and 4.1, 10.4, 7.7 or 5.1% with 2.5mm-fructose. 5. Fructose at 7.5 and 25mm decreased the 3H2O yields to less than the control values, but had no apparent effect on the amount of [U-14C]glucose metabolized. 6. In the incubations with 5mm-glucose and 25mm-fructose there were significant decreases in heat production, O2 consumption and in the ratio of O2 uptake to heat output. 7. Fructose at 2.5mm caused a 64% increase in heat output, but only a 43% increase in O2 uptake. 8. The radioisotopic and calorimetric data demonstrate that physiological concentrations of fructose greatly increase metabolism in hepatocytes from starved rats. These data also indicate increased cycling at glucose/glucose 6-phosphate and at fructose 6-phosphate/fructose 1,6-bisphosphate in the presence of 2.5mm-fructose, although the rates of cycling were actually decreased relative to the amount of glucose catabolized. 9. At concentrations of 2.5, 7.5 and 25mm, fructose depressed hepatocyte ATP concentrations by 20, 65 and 80% respectively. Although fructose at 7.5 and 25mm increased glucose and lactate release, O2 consumption, production of heat and formation of3H2O from [1-3H]-, [2-3H]-, [3-3H]- or [6-3H]-glucose were lowered to values equal to, or less than, controls. These effects probably reflect a severe derangement of hepatic metabolism due to excess phosphorylation of fructose when present at high concentrations.
Turnover of adenosine 3′:5′-cyclic monophosphate in chicken erythrocytes
Authors: Gorin E , Dickbuch S
Published: August 2015

Turnover of cyclic AMP was studied in intact chicken erythrocytes. Production of cyclic AMP was stimulated by adrenaline and then blocked by propranolol. The decline in the cyclic AMP concentration under these conditions is solely due to its intracellular degradation, whereas efflux of the nucleotide, although existing in these cells, does not contribute significantly to the change in its concentration. Intracellular degradation of cyclic AMP follows a first-order kinetics with a half-life of about 6 min. Similar half-lives were obtained at widely different adrenaline concentrations or when the ration of propranolol to adrenaline was varied by 25-fold. Theoretical equations were applied to calculate the rates of cyclic AMP synthesis and degradation in the intact cells under different experimental conditions. Maximal adrenaline concentrations raise the rate of cyclic AMP synthesis and its steady-state concentration by about 10-fold. The addition of caffeine causes a further 33% increase in intracellular concentration of the nucleotide, which is in good agreement with the theoretical increase computed from its slowed-down degradation.
Secretion of haem by hepatic parenchymal cells
Authors: Bissell D M , Liem H H , Muller-Eberhard U
Published: August 2015

Hepatic parenchymal cells in primary culture, and also the intact perfused liver, secrete newly synthesized haem into extracellular fluids. In cultures incubated with the haem precursor delta-amino[4-14C]laevulinate, labelled haem was formed at a linear rate for at least 8 h, and 10-20% of the total labelled haem was present in the culture medium. The appearance of labelled extracellular haem was proportional both to the concentration of labelled precursor offered to the cells and to the time of incubation. Similar results were obtained when [2-14C]glycine was added as haem precursor. Studies with the isolated perfused liver indicated that newly synthesized haem is secreted also by the intact liver. Approximately equal amounts of haem appeared in the bile and in perfusate. The findings are discussed in relation to the pathogenesis of symptoms in the hereditary hepatic porphyrias.
Binding of Ricinus communis agglutinin to the mitochondrial inner membrane as an artifact during preparation
Authors: Köhle H , Kauss H
Published: August 2015

Endosperm from Ricinus communis was homogenized in the presence of 3H-labelled Ricinus communis agglutinin, with or without addition of lactose. In preparations without the binding-specific sugar the subfraction containing the mitochondrial inner membrane contained sufficient labelled agglutinin to account for the agglutinin reported to be associated with this membrane.
Demonstration and cytosolic location of an endo-N-acetyl-β-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type substrate in rat liver
Authors: Pierce R J , Spik G , Montreuil J
Published: August 2015

An endo-N-acetyl-beta-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type glycoasparagine substrate was demonstrated in rat liver. This activity was optimal at pH 7.0 and was predominantly present in the soluble (cytosolic) fraction.
An accurate and sensitive analysis by high-pressure liquid chromatography of conjugated and unconjugated bilirubin IX-α in various biological fluids
Authors: Onishi S , Itoh S , Kawade N , Isobe K , Sugiyama S
Published: August 2015

An accurate and sensitive method was developed for the complete separation of the native tetrapyrroles, such as bilirubin and its mono- and di-conjugates of glucuronic acid, glucose and xylose, by ion-pair reversed-phase high-pressure liquid chromatography. The application of this method was demonstrated by the analysis of bile pigments in human bile and urine, and the method also makes it possible to estimate very low UDP-glucuronyltransferase activity, such as is found in the human foetal and neonatal liver.
A low-molecular-weight inhibitor of the neutral proteinase from rat intestinal smooth muscle
Authors: Carney I T , Curtis C G , Kay J K , Birket N
Published: August 2015

1. Rat intestinal smooth muscle was shown to contain endogenous inhibitory activity towards the neutral trypsin-like muscle proteinase described previously [Beynon & Kay (1978) Biochem. J. 173, 291-298]. 2. Comtamination of the muscle tissue by mucosal, blood and pancreatic inhibitors was shown to be unlikely. 3. The inhibitory activity was resolved into high- and low-molecular-weight components. 4. The low-molecular-weight component was purified to homogeneity. It has a molecular weight of approx. 9000 and was stable over the pH range 3-11. 5. It inhibited the muscle proteinase competitively (Ki congruent to t microM), but had no effect on any of the other proteinases tested. 6. Leupeptin also inhibited the muscle proteinase competitively (Ki congruent to 0.3 microM), whereas the low-molecular weight proteins gastrin, glucagon and insulin B-chain had very little effect. 7. A role for a weakly binding inhibitor in modulating the influence of the neutral proteinase on intracellular protein degradation is considered.
Activation and inactivation of phosphoenolpyruvate carboxykinase by ferrous ions
Authors: Reynolds C H
Published: August 2015

Phosphoenolpyruvate carboxykinase from rat liver cytosol is activated by Fe2+ ions in either direction of catalysis. Preincubation of the purified enzyme with Fe2+ ions causes a time-dependent irreversible loss of activity; this is not seen with unpurified enzyme. Purified enzyme can be protected from inactivation by Fe2+ ions by partially purified protein fractions from liver (ferroactivator fractions). The possible role of ferroactivator and Fe2+ ions in regulating phosphoenolpyruvate carboxykinase is discussed.
The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity (reverse reaction)
Authors: Cummings J , Graham A B , Wood G C
Published: August 2015

Arrhenius plots of the non-latent UDP-glucuronlytransferase reverse reaction (p-nitrophenyl glucuronide donor) activity of guinea-pig microsomal membranes prepared with 15 mM-KCl were linear from 5 to 40 degrees C. These plots for other preparations from guinea-pig and rat liver (i.e. preparations that show transferase latency) exhibited two linear regions intersecting at a transition point near 19-21 degrees C. This discontinuity was abolished when latency was removed by treating the membranes with perturbants of phospholipid-bilayer structure. Thus the temperature-depdendnces of the reverse reaction catalysed by the enzymes of these various preparations are similar to those of the corresponding forward reactions [Pechey, Graham & Wood (1978) Biochem. J. 175, 115-1124]. Perturbants activated the enzyme of KCl-prepared guinea-pig microsomal membranes only slightly and caused no significant alteration to Arrhenius plots of its forward or reverse reaction activities. These results support the ‘compartmentation’ theory of UDP-glucuronyltransferase lactency.
The chemical composition of bovine vitreous-humour collagen fibres
Authors: Swann D A , Sotman S S
Published: August 2015

The insoluble protein fraction was prepared from the central and posterior peripheral fraction of bovine vitreous humour. The collagen present in this fraction was solubilized by pepsin and fractionated by gel chromatography. Analysis of the solubilized collagen fractions showed that the alpha-chain component had an amino acid composition and yielded a series of CNBr-cleavage peptides that showed it was very similar to type II collagen obtained from articular cartilage. Bovine vitreous-humour collagen alpha-chains differed, however, from those of cartilage collagen in that they had a lower alanine content and differed in their susceptibility to cleavage by CNBr. Satisfactory cleavage was obtained after two CNBr treatments involving reduction and alkylation. In addition, significant quantities of other peptides constituents were present in the vitreous-humour collagen fractions, and the galactose and glucose content of the alpha-chain fraction was more than double that of the same fraction obtained from articular cartilage. Although the origin of the additional peptide constituents in the vitreous-humour collagen preparations is not known, the results obtained indicate that they are probably not derived from a distinct type of alpha-chain component but may be terminal peptides covalently linked to the alpha 1 type-II helical portions of the collagen. The differences in the chemical composition of the vitreous-humour collagen indicate that vitreous-humour fibres are composed of a special type-II collagen.
The isolation and preliminary characterization of N-acetyl-D-glucosamine kinase from rat kidney and liver
Authors: Allen M B , Walker D G
Published: August 2015

1. Procedures for the extensive purification in high yield of N-acetyl-D-glucosamine kinase from rat liver and kidney are described. The separation of this enzyme from hepatic glucokinase depended primarily on their differing behaviour on an affinity column of Sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose. 2. This N-acetyl-D-glucosamine kinase also catalyses the phosphorylation of N-acetyl-D-mannosamine and, at a lower rate, several other sugar analogues, including D-glucose. 3. A comparison of the behaviour of the enzyme during gel filtration and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels suggests that N-acetyl-D-glucosamine kinase is a symmetrical dimer of mol.wt. 80000.
Kinetic characterization of N-acetyl-D-glucosamine kinase from rat liver and kidney
Authors: Allen M B , Walker D G
Published: August 2015

1. Under normal assay conditions the N-acetyl-D-glucosamine kinases from rat liver and kidney show a pH-dependent lag phase before reaching a steady state, which is probably due to reversible dissociation of the dimeric enzyme. 2. The enzyme catalyses the phosphorylation of N-acetyl-D-glucosamine, N-acetyl-D-mannosamine and D-glucose at pH 7.5, with apparent Km values of 0.06, 0.95 and 600 mM respectively for the enzyme from liver and 0.04, 1.0 and 410 mM respectively for the kidney enzyme. It is strongly inhibited by ADP. 3. The interaction between the enzymes and acceptor substrates shows non-Michaelian kinetics with respect to N-acetyl-D-glucosamine but normal behaviour towards N-acetyl-D-mannosamine and D-glucose. 4. Both N-acetyl-D-glucosamine and N-acetyl-D-mannosamine inhibit the phosphorylation of D-glucose; this inhibition appears to be mixed in character. 5. The facts that the enzymes catalyse the phosphorylation of N-acetyl-D-mannosamine and D-glucose do not detract from the designation of the enzymes as N-acetyl-D-glucosamine kinase. Phosphorylation of glucose in vivo by these kinases is unlikely.
Properties of freshly purified and thiol-treated spinach chloroplast fructose bisphosphatase
Authors: Charles S A , Halliwell B
Published: August 2015

Freshly purified spinach chloroplast fructose bisphosphatase is powerfully inhibited by inorganic phosphate competitively with respect to its substrate fructose 1,6-bisphosphate. The concentrations of phosphate and substrate in the chloroplast stroma are such that the enzyme in this form could not operate at a significant rate in vivo. Incubation of the enzyme with dithiothreitol for 24 h decreases the Km for fructose 1,6-bisphosphate from 0.8 to 0.033 mM, decreases the Km for Mg2+ from 9 to 2 mM and substantially alleviates inhibition by inorganic phosphate. The physiological significance of thiol activation of the enzyme is discussed.
Net hepatic and splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in relation to lactation and nutrient supply
Authors: Baird G D , Lomax M A , Symonds H W , Shaw S R
Published: August 2015

1. Circulating concentrations of glucose, propionate, lactate and pyruvate, and net exchange of these compounds across the liver and gut, were measured in lactating and non-lactating dairy cows (a) in the normal fed state, (b) before, during and after intravenous infusion of an aqueous solution of glucose, propionate or lactate (lactating cows only) in fed animals, and (c) before and during 6 days of food deprivation. 2. In the normal fed state, gut output of propionate, hepatic output of glucose and hepatic uptake of lactate were all higher in the lactating group. There was a net uptake of pyruvate across the liver in the lactating cows and a net output in the non-lactating cows. In the lactating cows there was a net uptake of lactate and pyruvate by the splanchnic bed (i.e. gut and liver combined). 3. In the lactating cows, the glucose and propionate infusions had the following effects: decrease in net hepatic uptake of lactate; a switch in pyruvate exchange across the liver from uptake to output; suppression of uptake of lactate and pyruvate by the splanchnic bed; increase in the magnitude of the liver (propionate uptake)/(glucose output) ratio. Lactate infusion did not affect hepatic propionate uptake. 4. Food deprivation increased hepatic extraction of lactate and pyruvate and decreased the liver (propionate uptake)/(glucose output) ratio in both groups. 5. It is concluded that mechanisms exist to ensure an inverse relationship between the availability to the cow of glucose or propionate and utilization by the splanchnic bed of endogenously derived lactate and pyruvate.
Subcellular distribution and movement of 5'-nucleotidase in rat cells
Authors: Stanley K K , Edwards M R , Luzio J P
Published: August 2015

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.
An analysis of some thermodynamic properties of iron-sulphur centres in site I of mitochondria
Authors: Ingledew W J , Ohnishi T
Published: August 2015

1. The midpoint potentials of the various iron-sulphur centres in Site I were determined at different pH values by the technique of redox potentiometry. An interesting feature is the pH-dependence of Centre N-2, the highest potential component of the NADH dehydrogenase segment of the respiratory chain. 2. The apparent midpoint potentials of Centre N-2 (NADH dehydrogenase) and S-1 (succinate dehydrogenase) and their pH-dependence was also determined by using the succinate/fumarate couple. Again Centre N-2 is pH-dependent in midpoint potential, and Centre S-1 is not. The results obtained by titrating with the succinate/fumarate couple are in quantitative agreement with those obtained for these centres by redox potentiometry. 3. Oxidation-reduction titrations of iron-sulphur centres with the couple NADH/NAD+ and an analogue APADH/APAD+ in the presence of rotenone gave results substantially different from those obtained by redox potentiometry; these differences may be due to the mechanism of action of NADH dehydrogenase and its specific interaction with NADH. 5. The addition of ATP to an NAD+/NADH-poised system induces an uncoupler-sensitive oxidation of Centre N-4.
A simple and fast method for the isolation of basolateral plasma membranes from rat small-intestinal epithelial cells
Authors: Scalera V , Storelli C , Storelli-Joss C , Haase W , Murer H
Published: August 2015

A method was developed for the analytical and preparative isolation of basolateral plasma membranes from rat small intestine. They were separated on a self-orientating Percoll (modified colloidal silica) gradient starting with a heavy microsomal-membrane fraction and involving centrifugation at 48,000 g for 1 h. (Na+ + K+)-stimulated ATPase activity, used as a marker enzyme for the basolateral plasma membrane, is enriched 20-fold compared with that found in the homogenate of isolated intestinal epithelial cells.
The stimulus–secretion coupling of glucose-induced insulin release. Metabolism of glucose in K+-deprived islets
Authors: Sener Abdullah , Kawazu Shoji , Malaisse Willy J.
Published: August 2015

1. When pancreatic islets were exposed to a K+-free medium, the intracellular concentration of K+ was decreased and that of Na+ increased. 2. In the K+-deprived islets, the utilization of [5-3H]glucose, output of lactic acid and oxidation of [U-14C]-glucose were decreased by about 30–40% below the control values found at normal extracellular K+ concentration (5.0mm). However, the oxidation of [U-14C]pyruvate was unaffected. 3. The omission of extracellular K+ little affected the production of 14CO2 from islets prelabelled with [U-14C]palmitate and incubated in the absence of glucose, despite the fact that K+ deprivation significantly increased the ATP concentration and ATP/ADP concentration ratio in the glucose-deprived islets. 4. At normal K+ concentration, glucose increased the concentrations of phosphoenolpyruvate, NAD(P)H and ATP in the islets. In the glucose-stimulated islets, the concentration of phosphoenolpyruvate, but not that of either NAD(P)H or ATP, was higher in the absence than in the presence of extracellular K+. In islet homogenates, the activity of pyruvate kinase (EC 2.7.1.40) was stimulated by K+ (optimal activity at 100–150mm-K+) and inhibited by Na+ (except at very low K+ concentrations). 5. K+ could be replaced by NH4+, Rb+, Cs+ or Na+ to maintain, at least to some extent, pyruvate kinase activity in islet homogenates. Addition of Rb+ or Cs+, but not NH4+, to K+-deprived media also increased [U-14C]glucose oxidation by intact islets. 6. The omission of K+ did not cause any obvious anomaly in the apparent dependency of 45Ca2+ net uptake on NAD(P)H concentration in the islets. 7. These data suggest that the coupling between metabolic and ionic events in the islet cells involves feedback mechanisms through which glucose oxidation may be modulated by cationic factors.
Surface-labelling studies on skeletal-muscle cells in vitro. Heterogeneity of iodinated cell-surface proteins
Authors: Cates G A , Holland P C
Published: August 2015

1. Two distinct classes of protein were detected at the surface of chick-embryo skeletal-muscle cells after iodination of the cells in monolayer culture. 2. The two classes of iodinated proteins differed in their ability to co-purify with a vesicular plasma-membrane fraction prepared from surface-labelled cells. 3. One class consisted of predominantly high-molecular-weight glycoproteins that co-purified with the plasma-membrane fraction, but showed no significant qualitative or quantitative alterations in labelling with 125I and lactoperoxidase during myogenesis. 4. A second class of predominantly lower-molecular-weight proteins showed reproducible quantitative alterations in 125I-labelling during myogenesis but failed to co-purify with the plasma-membrane fraction. 5. This second class of proteins may represent matrix proteins involved in intercellular adhesion or adhesion of cells to the substratum. They are unlikely to be directly required for the process of plasma-membrane fusion during myogenesis, since they do not copurify with a vesicular plasma-membrane fraction known to be capable of Ca2+-dependent fusion in vitro.
Nature of the effect of adenosine 5'-monophosphate on the cyanide-insensitive respiration in mitochondria of Moniliella tomentosa
Authors: Vanderleyden J , Van Den Eynde E , Verachtert H
Published: August 2015

The alternative oxidase of Moniliella tomentosa mitochondria is stimulated by 5'-AMP. This effect may be masked, depending on the isolation procedure of the mitochondria. The preparation of submitochondrial particles results in the expression of the 5'-AMP effect. Two more methods are now described to reveal the 5'-AMP effect whenever it would be masked: (1) switching on the myokinase activity of the mitochondria to deplete them of endogenous 5'-AMP; (2) using detergents (sodium dodecyl sulphate, sodium deoxycholate) in a controlled detergent:protein ratio, or chloroform. The alternative oxidase of detergent-solubilized mitochondria was somewhat less selective towards nucleotides than were intact mitochondria. The effect of nucleotides on quinol oxidation by mitochondrial preparations and on quinol autoxidation was also studied. Mitochondrial oxidation of succinate by the alternative oxidase and autoxidation of quinols behaved similarly in the presence of certain nucleotides. Both reactions were stimulated. Both reactions were also inhibited by salicylhydroxamic acid. These effects on quinol oxidation disappeared when bovine serum albumin or mitochondrial proteins were present. From the results obtained it is not possible to exclude quinol autoxidation as a final step of the alternative oxidase.
Differences in the metabolism of very-low-density lipoproteins by isolated beating-heart cells and the isolated perfused rat heart. Evidence for collagenase-released extracellular lipoprotein lipase
Authors: Rajaram O V , Clark M G , Barter P J
Published: August 2015

1. The metabolism of VLD lipoproteins (very-low-density lipoproteins) was studied in intact isolated beating-heart cells and isolated perfused rat heart from starved animals by using [14C]triacylglycerol fatty acid-labelled VLD lipoprotein prepared from rats previously injected with [1-14C]palmitate. 2. 14C-labelled VLD lipoprotein was metabolized by the isolated perfused heart, but was only minimally metabolized by the heart cells unless an exogenous source of lipoprotein lipase was added. 3. Measurements of lipoprotein lipase at pH 7.4 with the natural substrate 14C-labelled VLD lipoprotein indicated that during collagenase perfusion of the heart the enzyme was released into the perfusate, the activity released being proportional to the concentration of collagenase used. Lipoprotein lipase activity in homogenates of hearts that had been perfused with collagenase showed a corresponding loss of activity. 4. At high perfusate concentrations of collagenase, inactivation of the released lipoprotein lipase occurred. 5. Lipoprotein lipase activity was largely undetectable in the homogenate of the isolated heart cells. 6. It is concluded that the lipoprotein lipase responsible for the hydrolysis of VLD lipoprotein triacylglycerol is predominantly located externally to the heart muscle cells and that its release can be facilitated by perfusion of the heart with bacterial collagenase.
Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration
Authors: Rachamin Gloria , Macdonald J. Alain , Wahid Samina , Clapp Jeremy J. , Khanna Jatinder M. , Israel Yedy
Published: August 2015

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these animals.
The effects of trypsin and phospholipase C on insulin binding and action in the isolated adipocyte
Authors: Clark S , Larkins R G , De Luise M , Melick R A
Published: August 2015

The effect of alterations to the insulin receptor on the insulin sensitivity of isolated adipocytes was studied. Receptor changes were induced by treatment of adipocytes with either phospholipase C or trypsin. After enzyme treatment, binding of insulin to insulin receptors and insulin-mediated glucose metabolism were examined. Exposure of adipocytes to phospholipase C (2 units/ml) significantly increased insulin binding to the cells, but destroyed the ability of the cells to oxidize glucose. After treatment with trypsin (500 micrograms/ml) for 5 min, insulin binding to the adipocytes was significantly increased. This was shown to be due to an increase in insulin-receptor affinity. Metabolic studies showed that trypsin treatment led to an increase in basal glucose transport but markedly decreased the response to insulin at all concentrations tested. Adipocytes treated with trypsin showed no significant difference in basal glucose oxidation rates when compared with controls, but were less sensitive to insulin at low insulin concentrations, and showed a decreased maximum response at high insulin concentrations. In conclusion, these findings indicate a dissociation between induced changes in binding of insulin to insulin receptors and subsequent hormone action. The importance of post-receptor events in the biological action of insulin is highlighted.
Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro
Authors: Kirby Christopher , Clarke Jacqui , Gregoriadis Gregory
Published: August 2015

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.
Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase
Authors: Houslay Miles D. , Dipple Irene , Elliott Keith R. F.
Published: August 2015

1. GTP, but not p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, 125I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and 125I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.
Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins
Authors: Cox R A , Greenwell P
Published: August 2015

It was shown that high-salt (2.75 M-NH4Cl/69mM-MgCl2) shock treatment at 0 degrees C of the larger subparticles (L-subparticles) of rabbit, Xenopus laevis and Neurospora crassa cytoplasmic ribosomes yielded split-protein fractions that were not only functionally equivalent but also interchangeable. Thus, although the remaining core-particles were inactive in both the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis, activity was restored when they were combined with either homologous or heterologous split-protein fractions. This technique was used to prepare active hybrid L-subparticles, e.g. rabbit cores/N. crassa split-proteins, and also active hybrid ribosomes, e.g. rabbit smaller subparticle/X. laevis core-particle/rabbit split-proteins. Rabbit and X. laevis split-protein fractions labelled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride were both shown to bind to rabbit core-particles in approximate correlation with the degree of re-activation. The split-protein fractions of rabbit and X. laevis L-subparticles were analysed by two-dimensional and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights (measured in sodium dodecyl sulphate gels) of the split-proteins of rabbit and X. laevis L-subparticles were found to be similar. These results demonstrate that the peptidyltransferase active centre of cytoplasmic ribosomes of eukaryotes has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active centre is highly conserved.
Utilization of membranous lipid substrates by membranous enzymes. Hydrolysis of sphingomyelin in erythrocyte ‘ghosts’ and liposomes by the membranous sphingomyelinase of chicken erythrocyte ‘ghosts’
Authors: Record M , Loyter A , Gatt S
Published: August 2015

Incubation at 37 degrees C of haemolysed chicken erythrocytes (‘chicken erythrocyte ghosts’) resulted in hydrolysis of the membrane sphingomyelin, suggesting an activation of a latent sphingomyelinase during the haemolysis procedure. When this incubation was continued for several hours, the entire sphingomyelin of the erythrocyte ‘ghosts’ was hydrolysed and membranes were obtained that were devoid of sphingomyelin, but had an active sphingomyelinase. Mixing the latter membranes with human erythrocyte ‘ghosts’ or positively charged liposomes led to hydrolysis of the sphingomyelin in these two membranes. This suggested that, after haemolysis, the activated sphingomyelinase in the membrane of the chicken erythrocyte ‘ghosts’ could hydrolyse sphingomyelin in its own membrane (‘intramembrane utilization’) or adjacent membranes (‘intermembrane utilization’).
Primary structure of the calcium ion-transporting adenosine triphosphatase from rabbit skeletal sarcoplasmic reticulum. Some peptic, thermolytic, tryptic and staphylococcal-proteinase peptides
Authors: Allen G , Bottomley R C , Trinnaman B J
Published: August 2015

The soluble peptides from the peptic digest of the reduced S-carboxymethylated 3-carboxypropionylated adenosine triphosphatase protein have been isolated and most of their structures have been determined. About 397 residues of the protein were represented in these peptides. The reduced S-carboxymethylated protein was digested with thermolysin, and peptides containing arginine or carboxymethylcysteine were isolated and characterized. Some peptides isolated from tryptic and staphylococcal-proteinase digests of the protein are described. The information contained within the structures of these peptides has been used to reconstruct long stretches of the sequence of the ATPase protein that constitute most of the protein structure external to the lipid bilayer (Allen, Trinnaman and Green (1980) Biochem. J. 187, 591-616). The details of some of the chromatographic steps used in the isolation of the peptides and the properties of the peptides are contained in Supplementary Publication SUP 50104 (45 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
The primary structure of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum. Peptides derived from digestion with cyanogen bromide, and the sequences of three long extramembranous segments
Authors: Allen G , Trinnaman B J , Green N M
Published: August 2015

The isolation and characterization of the soluble peptides from the CNBr digest of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum are described. The 562 unique residues of the protein were placed in sequences. The remaining part of the protein (about 500 residues) yielded long hydrophobic sequences that contained all but one of the tryptophan residues of the protein and that were probably derived largely from the intramembranous parts of the protein. Three long stretches of primary structure, constituting half of the protein, have been reconstructed from the information presented here together with the sequences found in peptides from other digests of the protein. The secondary structures of these sequences have been predicted. A model for the primary structure of the protein is presented and the implications discussed. Details of the isolation of peptides are contained in Supplementary Publication, SUP 50105 (29 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
Characterization of the intermediates in the reaction of membrane-bound mixed-valence-state cytochrome oxidase with oxygen at low temperatures by optical spectroscopy in the visible region
Authors: Clore G M
Published: August 2015

The ‘pure’ difference spectra of the three species, IM, IIM and IIIM, formed in the low-temperature reaction of membrane-bound mixed-valence-state cytochrome oxidase with O2 relative to unliganded membrane-bound mixed-valence-state cytochrome oxidase were characterized by optical spectroscopy in the visible region. The difference spectrum of species IM was characterized by a peak at 590 nm and a trough at 608 nm, that of species IIM by a peak at 606 nm, and that of species IIIM by a peak at 610 nm. A comparison with the difference spectra of species IIM and IIIM obtained with soluble cytochrome oxidase [Clore, Andréasson, Karlsson, Aasa & Malmström (1980) Biochem. J. 185, 155-167] revealed small but significant differences in the peak positions and bandwidths of the 605-610 nm absorption band.
Antibody combining sites can be mimicked synthetically. Surface-simulation synthesis of the phosphorylcholine-combining site of myeloma protein M-603
Authors: Kazim A L , Atassi M Z
Published: August 2015

By applying the concept of ‘surface-simulation’ synthesis to the combining site of the myeloma protein M-603 we were able to mimic synthetically its phosphorylcholine-binding characteristics. The synthetic surface-simulation peptide was found to bind to phosphorylcholine, whereas a control peptide that had the same amino acid composition but a different sequence showed little or no binding activity. The specificity of the binding was further confirmed by inhibition studies in which the surface-simulation peptide, but not the control peptide, inhibited the binding of 125I-labelled surface-simulation peptide to phosphorylcholine. Furthermore, the surface-simulation peptide was found to completely inhibit the binding of the native myeloma protein, M-603, to phosphorylcholine. The control peptide was unable to inhibit this binding. These findings suggest that surface-simulation synthesis can be effectively employed to mimic synthetically antibody combining sites, and may in the future be a valuable tool with which to manipulate the immune response to clinically important antigens.