bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermodynamics of porphyrin dimerization in aqueous solutions
Authors: Margalit R , Rotenberg M
Published: August 2015

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 × 10(6)M-1 and 5.4 × 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van't Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = −36. 4kJ X mol-1; delta H0 = −46. 0kJ X mol-1 and delta S0 = −32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the ‘ solvophobic force’ theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.
Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat β-cell tumour
Authors: Tooke N E , Hales C N , Hutton J C
Published: August 2015

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.
Multiple effects of short-chain alcohols on binding to rat heart muscarinic receptors
Authors: Waelbroeck M , Robberecht P , De Neef P , Christophe J
Published: August 2015

Short-chain alcohols inhibited the equilibrium binding of agonists and antagonists to rat heart muscarinic receptors. Methanol, ethanol, propan-2-ol and propan-1-ol, when used at low concentrations, behaved as pseudo-competitive antagonists. Their rank order of potency paralleled their relative partition coefficients, suggesting that this inhibition was simply due to the interaction of the alcohols with a hydrophobic part of the receptor or with membrane lipids. The four alcohols increased the dissociation rate constant of [3H]oxotremorine M from the high-affinity agonist receptors and decreased the stability of this receptor state. These effects might reflect increased membrane fluidity and/or decreased hydrophobic interactions (see below). By contrast, the effects of alcohols on the association and dissociation rates of N-[3H]- methscopolamine (an antagonist) were not correlated to their relative octanol/water partition coefficient (a measure of their affinity for biophases). Alcohols, at the relatively high concentrations necessary for increased membrane ‘fluidity’, are known to affect the relative stability of various protein conformations. We believe that the effects of alcohols on antagonist binding to rat heart muscarinic receptors reflected changes in the activation energy of association and dissociation reactions, the inhibition of equilibrium binding being mainly due to decreased ‘hydrophobic interactions’.
The effect of mitogenic lectins and monoclonal antibodies on intracellular free calcium concentration in human T-lymphocytes
Authors: O'Flynn K , Linch D C , Tatham P E R
Published: August 2015

The effect of mitogenic lectins and the mitogenic antibody UCHT1 on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in human lymphocytes was investigated by using the fluorescent Ca2+-indicator quin2 . Phytohaemagglutinin, concanavalin A and UCHT1 increased [Ca2+]i in T-cells to a maximum level within 2 min. No T-cell response was seen with poke weed mitogen. None of the mitogens affected non-T-cell [Ca2+]i.
Purification and characterization of human neuropeptide Y from adrenal-medullary phaeochromocytoma tissue
Authors: Corder R , Emson P C , Lowry P J
Published: August 2015

Human neuropeptide Y was isolated from acid extracts of adrenal-medullary phaeochromocytoma tissue. After (NH4)2SO4 fractionation, the neuropeptide Y-like immunoreactivity was purified from the resolubilized 80%-saturation-(NH4)2SO4 peptide-rich precipitate, by gel filtration, cation-exchange chromatography and reverse-phase high-pressure liquid chromatography. Amino acid analysis of the peptide revealed a composition almost identical with that of the pig peptide, the exception being the loss of one leucine residue and its replacement with methionine. Tryptic digestion of the peptide and subsequent amino acid analysis of the fragments further confirmed the identity of the peptide. Carboxypeptidase Y digestion of the (1-19)-peptide tryptic fragment has shown the methionine to be located at position 17 in human neuropeptide Y.
Glycosidases induced in Aspergillus tamarii. Secreted α-d-galactosidase and β-d-mannanase
Authors: Civas A , Eberhard R , Le Dizet P , Petek F
Published: August 2015

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.
Metabolic activation of acetylenes. Covalent binding of [1,2-14C]octyne to protein, DNA and haem in vitro and the protective effects of certain thiol compounds
Authors: White I N H , Campbell J B , Farmer P B , Bailey E , Nam N H , Thang D C
Published: August 2015

[1,2-14C]Oct-l-yne was used to investigate metabolic activation of the ethynyl substituent in vitro. Activation of octyne by liver microsomal cytochrome P-450-dependent enzymes gave intermediate(s) that bound covalently to protein, DNA and to haem. The time course and extent of covalent binding of octyne to haem and to protein were similar. However, two different activating mechanisms are probably involved. Whereas covalent binding to protein or to DNA was inhibited by nucleophiles such as N-acetylcysteine, that to haem was little affected. When N-acetylcysteine was included in the reaction mixtures, two major octyne-N-acetylcysteine adducts were isolated and purified by high-pressure liquid chromatography. G.l.c.-mass spectrometry and n.m.r. suggest that these are the cis-trans isomers of S-3-oxo-oct-1-enyl-N-acetylcysteine. Oct-1-yn-3-one reacted non-enzymically with N-acetylcysteine at pH 7.4 and 37 degrees C with a t1/2 of about 6 s also to yield S-3-oxo-oct-l-enyl-N-acetylcysteine. The same product was formed when microsomal fractions were incubated with oct-1-yn-3-ol, N-acetylcysteine and NAD(P)+. Octyn-3-one did not appear to react with haem or protoporphyrin IX. 5. A mechanism for the metabolic activation of oct-1-yne is proposed, consisting in (a) microsomal hydroxylation of the carbon atom alpha to the acetylenic bond and (b) oxidation to yield octyn-3-one as the reactive species.
Glucocorticoids and catecholamines as mediators of acute-phase proteins, especially rat α-macrofoetoprotein
Authors: van Gool J , Boers W , Sala M , Ladiges N C J J
Published: August 2015

Adrenal hormones were studied as possible triggering substances of the synthesis of acute-phase reactants in rats. alpha-Macrofoetoprotein, which rises sharply in concentration during inflammation, was used to monitor the acute-phase reaction. In normal rats glucocorticoids and catecholamines induce alpha-macrofoetoprotein synthesis; glucocorticoids only increase alpha-macrofoetoprotein to moderate levels in plasma, but catecholamines enhance alpha-macrofoetoprotein synthesis to very high levels, comparable with those observed in the post-injury phase. However, catecholamines in vivo also activate the adrenal cortex, suggesting a synergistic effect of both kinds of adrenal hormones. Our study showed that in adrenalectomized rats, the effect of catecholamines on alpha-macrofoetoprotein synthesis is greatly diminished, whereas the moderate effect of glucocorticoids remains. Combination of glucocorticoids and catecholamines induces extremely high alpha-macrofoetoprotein levels in both adrenalectomized and normal rats. With crossed immunoelectrophoresis it was shown that other acute-phase reactants, such as haptoglobin and alpha 1-major acute-phase protein, are affected differently by the hormones. Contrary to glucocorticoids, catecholamines give a pattern comparable with that found after surgical injury.
The extraction and mechanism of a novel isomaltulose-synthesizing enzyme from Erwinia rhapontici
Authors: Cheetham P S J
Published: August 2015

The single enzyme that mediates the bioconversion is demonstrated to be located in the cells' periplasmic space, a site that facilitates its use as an industrial biocatalyst, and to be a previously undescribed hexosyltransferase with four novel features. The enzyme is sucrose-specific, and has an intramolecular mechanism in which both glucose and fructose residues appear to be enzyme-bound. Thirdly, it is reaction-non-selective, forming simultaneously isomaltulose and a second hitherto uncharacterized alpha-(1----1)-linked disaccharide (trehalulose), by hydrolysis of sucrose followed by reaction of glucose with the C-6 and C-1 positions of the fructofuranose respectively. Finally, on extended incubation an unusual recycling mechanism caused the concentration of isomaltulose, the kinetically preferred product, to reach a transient maximum concentration and then fall, and the concentration of trehalulose, the thermodynamically favoured product, to rise slowly.
Influence of phosphatidylserine on (Na+ + K+)-stimulated ATPase and acetylcholinesterase activities of dog brain synaptosomal plasma membranes
Authors: Tsakiris S , Deliconstantinos G
Published: August 2015

Phosphatidylserine (PtdSer) incubated with synaptosomal plasma membranes (SPM) of dog brain is incorporated into SPM in proportion to its concentration in the incubation medium. Low PtdSer concentrations progressively activated the SPM-associated (Na+ + K+)-stimulated ATPase and acetylcholinesterase. Increasing the PtdSer concentration above that which maximally stimulated the enzyme activities effected a progressive inhibition with respect to maximal stimulation. Arrhenius plots of (Na+ + K+ + Mg2+)-dependent ATPase and 5′-nucleotidase revealed a clear break at 23-24 degrees C for both enzymes in SPM untreated with PtdSer (controls), whereas a linear relation was obtained for SPM treated with PtdSer. Changes in the allosteric properties of (Na+ + K+)-stimulated ATPase by fluoride (F-) and/or of 5′-nucleotidase by concanavalin A (i.e. changes of Hill coefficients) indicate that PtdSer increases the membrane fluidity. These results suggest that modifications of lipid-protein interactions in SPM induced by PtdSer may have implications in the physiological processes in the central nervous system.
Glycogenolytic effects of the calcium ionophore A23187, but not of vasopressin or angiotensin, in foetal-rat hepatocytes
Authors: Freemark M , Handwerger S
Published: August 2015

Vasopressin, angiotensin and phenylephrine stimulate glycogenolysis in postnatal rat liver by a Ca2+-mediated mechanism not involving cyclic AMP. To determine whether these hormones promote glycogenolysis in foetal liver, we have examined their effects, and those of the Ca2+ ionophore A23187, on glycogen metabolism in cultured foetal-rat hepatocytes. Vasopressin and angiotensin (0.1 nM-0.1 microM) had no effects on either glycogen synthesis (as assessed by [14C]glucose incorporation into glycogen) or phosphorylase a activity. However, A23187 at 1 and 10 microM inhibited glycogen synthesis by 31.3 and 89.1% respectively (both P less than 0.001) and stimulated phosphorylase a activity by 66.9 and 184.1% respectively (both P less than 0.01). Incubation of cells in Ca2+-deficient medium attenuated the effects of 10 microM-A23187 on glycogen synthesis and abolished the effects of 1 microM-A23187. As in postnatal liver, glucagon (1 and 20 nM) and isoprenaline (1 and 10 microM), which activate adenylate cyclase, inhibited glycogen synthesis and stimulated phosphorylase a activity in foetal hepatocytes. The minimal effective concentration of phenylephrine was 10 times that of isoprenaline. These results indicate striking differences in the ontogeny of cyclic AMP-mediated and Ca2+-mediated processes which regulate hepatic glycogenolysis. Since increases in cytosolic Ca2+ induce glycogenolysis in foetal-rat liver, the weak or absent responses to vasopressin, angiotensin and the alpha-adrenergic agonists may result from defects in hormone-receptor binding or in post-receptor events leading to the mobilization of intracellular Ca2+ stores.
Hyaluronate is synthesized at plasma membranes
Authors: Prehm P
Published: August 2015

The hybrid cell B6 line, which synthesizes large amounts of hyaluronate as the predominant glycosaminoglycan, was grown in the presence of [3H]glucosamine. The [3H]hyaluronate has a high molecular weight and was excluded by Sephacryl S-1000. After disruption of the cells the [3H]hyaluronate could further be elongated by incubation with UDP-GlcNAc and UDP-[14C]GlcA, yielding a hybrid molecule of hyaluronate labelled with [3H]GlcNAc and [14C]GlcA. Treatment of the cells with hyaluronidase before disruption eliminated the large [3H]hyaluronate and elongation of nascent chains in vitro commenced from low-molecular-weight chains. Thus nascent hyaluronate chains were degraded extracellularly by hyaluronidase and were therefore synthesized at the inner side of plasma membranes and extruded to the cell surface.
The influence of glucocorticoid on the fibrinogen messenger RNA content of rat liver in vivo and in hepatocyte suspension culture
Authors: Princen H M G , Moshage H J , de Haard H J W , van Gemert P J , Yap S H
Published: August 2015

The plasma concentration of fibrinogen, one of the major acute-phase proteins produced by the liver, increases during the acute-phase response as a result of enhanced synthesis in liver. Since adrenal-cortical hormones have been thought to have a key role in the regulation of the fibrinogen synthesis, fibrinogen-polypeptide mRNA sequences were determined in the present study, by using a specific complementary-DNA probe, in RNA fractions obtained from rat hepatocytes exposed to glucocorticoids in vitro (hepatocyte suspension cultures) and in vivo. Maximal induction of the fibrinogen-polypeptide mRNA (to 400% of the control value) was found in vitro at 0.1 microM-dexamethasone after 9 h of incubation. The same magnitude of induction was obtained with 20 microM-cortisol or 60 microM-corticosterone. In contrast with the findings in vitro, no induction of the fibrinogen-polypeptide mRNA was observed in the liver at various times after injection of different doses of glucocorticoids into rats. These results suggest that more complex regulatory mechanisms are involved and that glucocorticoids are not the sole regulatory factors in vivo in the enhanced synthesis of fibrinogen during the acute-phase response.
A cold-clamping technique for the rapid sampling of rat liver for studies on enzymes in separate cell fractions. Suitability for the study of enzymes regulated by reversible phosphorylation-dephosphorylation
Authors: Easom R A , Zammit V A
Published: August 2015

A technique for the rapid sampling, cooling and homogenization of rat liver is described. Its effectiveness in preserving the activity status of pyruvate kinase (soluble) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) (microsomal) during sampling is assessed in comparison with that of the freeze-clamping technique and of simple excision and mincing of liver tissue before homogenization. The results suggest that cold-clamping is equally effective as freeze-clamping in preserving the activity status of pyruvate kinase in liver samples obtained in situ, but in addition allows the subsequent separation of subcellular fractions, notably microsomes (microsomal fractions) and mitochondria. It is suggested that this property makes the technique useful in studying the activity status of enzymes (e.g. HMG-CoA reductase) the assay of which is subject to interference from the activity of other enzymes which are released from damaged organelles in crude homogenates of freeze-clamped liver samples. This suggestion was tested directly; the cold-clamping technique was found to preserve a substantially higher initial/total HMG-CoA reductase activity ratio [Easom & Zammit (1984) Biochem. J. 220, 739-745] in subsequently isolated microsomes compared with that obtained in microsomes prepared from liver samples processed in the conventional manner. The integrity of mitochondria isolated from homogenates of cold-clamped liver samples was preserved, as judged by the latency of intramitochondrial enzymes and by good respiratory control of the mitochondria. Possible further areas of metabolic studies to which the cold-clamping technique could be applied are suggested.
Membrane-potential-dependent changes of the lipid microviscosity of mitochondria and phospholipid vesicles
Authors: O'Shea P S , Feuerstein-Thelen S , Azzi A
Published: August 2015

The effects of a transmembrane potential difference upon the lipid microviscosity of cytochrome oxidase vesicles (COVs) and rat liver mitochondria (RLM) were investigated. COVs and RLM were labelled with the fluorescent probe 1,6-diphenylhexa-1,3,5-triene (DPH). The fluorescence polarization of the probe was then measured when potentials of different magnitudes were induced across the membranes of these particles. It was shown that the absolute value of the microviscosity changes to quite a significant extent, owing to the imposition of large membrane potentials. On relaxation of the membrane potential the lipid microviscosity was also shown to return to the value before the induction of the potential. The largest change in lipid microviscosity was observed when coupled respiration was initiated. This occurred in both the COV system and the RLM system. The absolute value of the lipid microviscosity was shown to change by as much as 22% with the induction of membrane potentials, owing to respiration. To confirm the viscosity measurements made with DPH, lipid microviscosity was also measured with the spin-labelled fatty acid 5-doxyl stearate. Measurements of the order parameters indicated that, in agreement with the results of fluorescence experiments, viscosity changes occurred that were due to the induction of a membrane potential. The significance of these findings to the regulation of metabolism is briefly discussed, the main conclusion being that, although there is certainly a significant variation of lipid microviscosity with electric field, mechanistic interpretations will require further studies.
The identification of threonine-95 as the major site of glycosylation in normal human myelin basic protein
Authors: Cruz T F , Wood D D , Moscarello M A
Published: August 2015

The enzymic transfer of N-acetylgalactosamine to myelin basic protein and that to peptides derived from basic protein were compared. Basic protein treated with pepsin and trypsin before glycosylation resulted in decreases of 7% and 23% in the amount of N-acetylgalactosamine transferred to the peptides respectively. However, digestion of basic protein had little effect on the sites glycosylated. It was found that Thr-95 was the major site for glycosylation in both the intact human basic protein and in the tryptic peptides.
Determination of cytochrome P-448 activity in biological tissues
Authors: Phillipson C E , Godden P M M , Lum P Y , Ioannides C , Parke D V
Published: August 2015

Three enzymes used for the determination of cytochrome P-448 activity, namely aryl hydrocarbon hydroxylase, biphenyl 2-hydroxylase and ethoxyresorufin O-de-ethylase, were evaluated with respect to their specificity, sensitivity and inducibility. Purified cytochrome P-448 (LM4), but not cytochrome P-450 (LM2), catalysed the O-de-ethylation of ethoxyresorufin in a reaction that was markedly inhibited by 9-hydroxyellipticine. After the administration of 3-methylcholanthrene to rats all three activities were induced, the extent of induction being highest for ethoxyresorufin O-de-ethylase. Administration of very small doses of benzo[a]pyrene (50 micrograms/kg) to rats to induce cytochrome P-448 specifically increased only the O-de-ethylation of ethoxyresorufin. 3-Hydroxybenzo[a]pyrene, the major metabolite determined by the aryl hydrocarbon hydroxylase assay, undergoes further NADPH-dependent oxygenation leading to loss of fluorescence. On the basis of these observations and those by other workers, we conclude that ethoxyresorufin O-de-ethylase provides the most specific, sensitive and reproducible means of determining cytochrome P-448 activity.
Volume-regulatory K+ efflux during concentrative uptake of alanine in isolated rat hepatocytes
Authors: Kristensen L ∅ , Folke M
Published: August 2015

Changes in cell volume and 42K+ efflux associated with concentrative alanine uptake were studied in isolated rat hepatocytes suspended in Krebs-Ringer bicarbonate buffer. After addition of 10 mM-alanine, cellular water volume increased by 15% and the rate constant of 42K+ efflux by 250%. Alanine-induced 42K+ efflux was abolished by quinine and was strongly decreased when the cell-volume increase was counteracted by sucrose. The results suggest that K+ efflux during alanine uptake is implicated in a volume-regulatory response.
The effects of insulin and glucagon on ketone-body turnover
Authors: Reed W D , Baab P J , Hawkins R L , Ozand P T
Published: August 2015

A double-isotope procedure was used to measure the effects of insulin and glucagon on ketone-body production and utilization (i.e. turnover) in the starved rat. Somatostatin was infused during the experiment to suppress the pancreatic release of either hormone. The immediate action of insulin in terms of ketone-body turnover that was most evident was a decreased production of 3-hydroxybutyrate, with no significant change in the turnover of acetoacetate. Similarly, the significant effect of glucagon infusion was to increase the production of 3-hydroxybutyrate, with minimum increase in acetoacetate turnover. The data support a direct effect of both hormones on the distribution of acetyl units derived from fatty acid beta-oxidation.
Loss of fast-twitch isomyosins in skeletal muscles of the diabetic rat
Authors: Rutschmann M , Dahlmann B , Reinauer H
Published: August 2015

By means of pyrophosphate electrophoresis the myosin isoenzyme pattern of two fast-twitch skeletal muscles (extensor digitorum longus, gastrocnemius) and one slow-twitch muscle (soleus) was investigated in control rats and was compared with that of rats 4 weeks after induction of diabetes mellitus by streptozotocin injection. In the fast-twitch muscles the isomyosin pattern consisting of FM1 (fast isomyosin 1), FM2 and FM3 was strongly affected by diabetes, resulting in an extensive loss of FM1 and a substantial decrease of FM2. These changes were also apparent when the light chains of the fast isomyosins were analysed by two-dimensional electrophoresis: LC3f (myosin light chain 3f) largely disappeared and LC2f was significantly diminished. In contrast, the isomyosin pattern in soleus muscle, consisting of SM1 (slow isomyosin 1) and SM2, was not affected by the diabetic state, and two-dimensional electrophoresis revealed a normal light-chain pattern of LC1sa, LC1sb and LC2s. These results indicate that the isomyosins of slow-twitch oxidative myofibres are more resistant to the hormonal and metabolic disorders during diabetes mellitus than are the isomyosins of fast-twitch fibres.
Calcium signals and phospholipid methylation in eukaryotic cells
Authors: Moore J P , Johannsson A , Hesketh T R , Smith G A , Metcalfe J C
Published: August 2015

Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.
The arsonomethyl analogue of adenosine 5′-phosphate. An uncoupler of adenylate kinase
Authors: Adams S R , Sparkes M J , Dixon H B F
Published: August 2015

Adenosine was converted into the arsonomethyl analogue of AMP. The reactions used provide a general route for converting an alcohol, R-CH2-OH, into the arsonomethyl analogue, R-CH2-CH2-AsO3H2, of its phosphate, R-CH2-O-PO3H2. The analogue of AMP proves to be a substrate for rabbit adenylate kinase, which shows a limiting velocity with it of 1/17 that with AMP, a Michaelis constant raised 70-fold to about 10 mM, and hence a specificity constant lowered about 1200-fold. The product of transfer of a phospho group from ATP to the analogue is, like all anhydrides of arsonic acids, unstable to hydrolysis, and so breaks down to yield orthophosphate and regenerate the analogue. Hence adenylate kinase is converted into an ATPase by the presence of the analogue.
Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon
Authors: Buechler K F , Beynen A C , Geelen M J H
Published: August 2015

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.
The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes
Authors: Hack N , Carey F , Crawford N
Published: August 2015

Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.
Effects of monensin on metabolism of isolated rat islets of Langerhans
Authors: Smith J E , Howell S L
Published: August 2015

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.
The discovery of a rapidly metabolized polymeric tetraphosphate derivative of adenosine in perfused rat heart
Authors: Mowbray J , Hutchinson W L , Tibbs G R , Morris P G
Published: August 2015

The predicted presence in perfused rat hearts of a rapidly metabolized but hitherto unrecognized form of adenosine phosphate has been confirmed by specific radioactive labelling. The properties of the purified compound suggest that it is a heteropolymer of a small organic acid, phosphate and purine nucleoside in the proportions 1:4:1.
Composition of partially purified NADPH oxidase from pig neutrophils
Authors: Bellavite P , Jones O T G , Cross A R , Papini E , Rossi F
Published: August 2015

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.
Electrostatic screening stimulates rate-limiting steps in mitochondrial electron transport
Authors: Møller I M , Kay C J , Palmer J M
Published: August 2015

The effect of electrostatic screening of fixed negative charges on uncoupled mitochondrial electron transport was investigated with substrates with different charge and different sites of donation of electrons to the electron-transport chain of Jerusalem-artichoke (Helianthus tuberosus L.) mitochondria. Duroquinol (neutral substrate) was oxidized with a pH optimum of 7.6-7.8. The addition of cations caused a doubling of Vmax. (order of efficiency C3+ greater than C2+ greater than C+) through electrostatic screening, whereas the Km was unaffected. Screening stimulated (by 150%) the Vmax. for the oxidation of reduced cytochrome c (positive substrate; to O2), but in this case the Km doubled. The Vmax. of the oxidation of exogenous NADH (negative substrate) was also stimulated by screening when the acceptor was O2, but unaffected when duroquinone was the acceptor. In both cases, the Km for NADH was considerably decreased. The effect of screening on the Km for the different substrates can be explained by the changes in the effective concentration of substrate near the active site due to the lowering in the size of the surface potential. The effect of screening on the Vmax. of the different partial processes indicates that increasing the salt concentration of the medium enhances the maximal activity of cytochrome c oxidase. However, the results also point at the existence of other rate-limiting steps, which are affected by screening and may involve ubiquinone, in electron transport in plant mitochondria.
Extraction and purification of proteoglycans from mature bovine bone
Authors: Franzén A , Heinegård D
Published: August 2015

Proteoglycans were extracted in good yields from the mineralized matrix of ground bovine bone, by using a two-step extraction procedure. Proteoglycans (8% of total), not associated with the bone mineral, were extracted at − 20 degrees C with 4M-guanidinium chloride containing proteinase inhibitors. Proteoglycans associated with the mineral, which accounted for 60% of the total, were then solubilized when EDTA was added to the extraction solvent. They were fractionated and purified in the presence of 4M-guanidinium chloride by CsCl-density-gradient centrifugations followed by chromatography on Sepharose CL-4B. Further purification was obtained by chromatography on DEAE-cellulose and hydroxyapatite in the presence of 7 M-urea. Three populations of proteoglycans and additional glycosaminoglycan peptides were obtained. The molecular dimensions of both intact molecules and of their side chains as well as their amino acid composition were different, indicating that they represent separate molecular entities. The main proteoglycan self-aggregated in the absence of 4M-guanidinium chloride or 7 M-urea, a property that was abolished when the proteoglycan core protein was fragmented.
Catabolism and lability of S-adenosyl-l-methionine in rat liver extracts
Authors: Eloranta T O , Kajander E O
Published: August 2015

The fate of S-adenosyl-L-methionine was studied in rat liver extracts by analysing the distribution of radioactivity from labelled adenosylmethionine in decomposition products, which were separated from each other by chromatographic and electrophoretic means. Marked non-enzymic degradation to adenine, pentosylmethionine, methylthioadenosine and homoserine was evident at pH 6.9-7.8. Enzymic cleavage to methylthioadenosine was stoichiometric with the accumulation of spermidine and could be totally prevented by inhibiting S-adenosyl-L-methionine decarboxylase. The results rule out the existence of adenosylmethionine cyclotransferase in rat liver and indicate that only two quantitatively significant enzymic processes are involved in hepatic adenosylmethionine degradation. Excluding nonenzymic decomposition, more than 99% of adenosylmethionine is demethylated and exclusively catabolized further by S-adenosyl-L-homocysteine hydrolase. Less than 1% of adenosylmethionine is decarboxylated and immediately utilized totally for polyamine biosynthesis.
A modified perifused incubation system for isolated fat-cells. Investigation of intermediary metabolism by perifusion
Authors: Harper R D
Published: August 2015

Perifused fat-cells showed similar values for acylglycerol glycerol synthesis from glucose with insulin and for the effects of added palmitate to those in normal incubations and those reported in the literature. Fatty acid synthesis was lower in perifused cells compared with normal incubations, and there was a net release of fatty acids only with the perifused fat-cells. Hence fluxes of metabolites were different in the two incubation systems, and the perifusion system enables the investigation of the flux of metabolites under conditions which may more closely resemble those in vivo.
Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase
Authors: Wuytack F , Raeymaekers L , Verbist J , De Smedt H , Casteels R
Published: August 2015

Membrane fractions prepared from smooth muscle of the pig stomach (antral part) contain two Ca2+-dependent phosphoprotein intermediates belonging to different Ca2+-transport ATPases. These alkali-labile phosphoproteins can be separated by electrophoresis in acid medium. The 130 kDa phosphoprotein resembles a corresponding protein in the erythrocyte membrane, whereas the 100 kDa protein resembles that of the Ca2+-transport ATPase in sarcoplasmic reticulum from skeletal muscle. These resemblances are expressed in terms of Mr, reaction to La3+ and in a similar proteolytic degradation pattern. The presence of the calmodulin-stimulated ATPase in mixed membranes from smooth muscle is confirmed by its binding of calmodulin and antibodies against erythrocyte Ca2+-transport ATPase, whereas such binding does not occur with proteins present in the presumed endoplasmic reticulum from smooth muscle.
Electron-paramagnetic-resonance spectroscopy studies on the dissimilatory nitrate reductase from Pseudomonas aeruginosad
Authors: Godfrey C , Greenwood C , Thomson A J , Bray R C , George G N
Published: August 2015

Preparations of nitrate reductase in the resting state from Pseudomonas aeruginosa exhibit an Mo(V) e.p.r. signal. Progressive reduction of the enzyme results at first in the intensification and then in the disappearance of the signal. Three different species of Mo(V) were detected by e.p.r. These are the high-pH species (g1 = 1.9871; g2 = 1.9795; g3 = 1.9632) and nitrate and nitrite complexes of a low-pH species (respectively g1 = 2.0004; g2 = 1.9858; g3 = 1.9670; and g1 = 1.9975; g2 = 1.9848; g3 = 1.9652). These signals are closely analogous to those for the enzyme from Escherichia coli described by Vincent & Bray [(1978) Biochem. J. 171, 639-647]. Signals typical of iron-sulphur clusters were also detected. In the oxidized enzyme these are believed to arise from a [3Fe-4S] cluster (g = 2.01) and in the reduced enzyme from an unusual low-potential [4Fe-4S]+ cluster (g1 = 2.054; g2 = 1.952; g3 = 1.878). The iron-sulphur centres were also studied in a ‘high-catalytic-activity’ form of the enzyme. Reduction with Na2S2O4 resulted in the formation of a complex signal with g values at 2.054, 1.952, 1.928, 1.903 and 1.878. The signal could be deconvoluted by reductive titration of the enzyme into two species (g1 = 2.054; g2 = 1.952; g3 = 1.878; and g1 = 2.036; g2 = 1.928; g3 = 1.903). The degradation of a [4Fe-4S] into a [3Fe-4S] cluster in the enzyme is suggested by these studies, the process being dependent on the method used to purify the enzyme. The addition of nitrate to the reduced enzyme results in the oxidation of Mo(IV) to Mo(V) and of all the iron-sulphur centres.
Stereochemistry of 1-(4′-hydroxyphenyl)ethanol produced by hydroxylation of 4-ethylphenol by p-cresol methylhydroxylase
Authors: McIntire W , Hopper D J , Craig J C , Everhart E T , Webster R V , Causer M J , Singer T P
Published: August 2015

Enzymic hydroxylation of 4-ethylphenol by (a) Pseudomonas putida and (b) highly purified p-cresol methylhydroxylase gave optically active 1-(4′-hydroxyphenyl)-ethanol. The products were transformed into the phenolic methyl ethers and shown to contain 69.5% and 65.6%, respectively, of the (S)-(-)-isomer. The stereochemistry of the reaction is discussed in terms of three distinct steps occurring at the active site of the enzyme.
Ferrous ion-EDTA-stimulated phospholipid peroxidation. A reaction changing from alkoxyl-radical- to hydroxyl-radical-dependent initiation
Authors: Gutteridge J M C
Published: August 2015

The stimulatory effect of ferrous salts on the peroxidation of phospholipids can be enhanced by EDTA when the concentration of Fe2+ in the reaction is greater than that of EDTA. Hydroxyl-radical scavengers do not inhibit peroxidation until the concentrations of Fe2+ and EDTA in the reaction are equal. Lipid peroxidation is then substantially initiated by hydroxyl radicals derived from a Fenton-type reaction requiring hydrogen peroxide. Superoxide radicals appear to play some role in the formation of initiating species.
Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes
Authors: Burgess G M , Irvine R F , Berridge M J , McKinney J S , Putney J W
Published: August 2015

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.
Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation
Authors: Jensen P , Wilson M T , Aasa R , Malmström B G
Published: August 2015

The inhibition of cytochrome c oxidase by cyanide, starting either with the resting or the pulsed enzyme, was studied by rapid-freeze quenching followed by quantitative e.p.r. It is found that a partial reduction of cytochrome oxidase by transfer of 2 electron equivalents from ferrocytochrome c to cytochrome a and CuA will induce a transition from a closed to an open enzyme conformation, rendering the cytochrome a3-CuB site accessible for cyanide binding, possibly as a bridging ligand. A heterogeneity in the enzyme is observed in that an e.p.r. signal from the cytochrome a3 3+-HCN complex is only found in 20% of the molecules, whereas the remaining cyanide-bound a3-CuB sites are e.p.r.-silent.
Hormonal regulation of L-type pyruvate kinase in hepatocytes from phosphorylase kinase-deficient (gsd/gsd) rats
Authors: Clark M G , Patten G S , Clark D G
Published: August 2015

The hormonal regulation of L-type pyruvate kinase in hepatocytes from phosphorylase b kinase-deficient (gsd/gsd) rats was investigated. Adrenaline (10 microM) and glucagon (10 nM) each led to an inactivation and phosphorylation of pyruvate kinase. Dose-response curves for adrenaline-mediated inactivation of pyruvate kinase, phosphorylation of pyruvate kinase and the stimulation of gluconeogenesis from 1.8 mM-lactate were similar for hepatocytes from control and gsd/gsd rats. Time-course studies indicated that adrenaline-mediated inactivation and phosphorylation of pyruvate kinase proceeded more slowly in phosphorylase kinase-deficient hepatocytes than in control hepatocytes. The age-dependent change in the adrenergic control of pyruvate kinase was similar between control and phosphorylase kinase-deficient hepatocytes. Adrenaline, glucagon and noradrenaline activated the cyclic AMP-dependent protein kinase and inhibited pyruvate kinase in phosphorylase kinase-deficient hepatocytes. Vasopressin (0.2-2 nM), angiotensin (10nM) and A23187 (10 microM) had no effect on the activity ratio of the cyclic AMP-dependent protein kinase or pyruvate kinase in these cells. It is concluded that phosphorylase kinase plays no significant role in the hormonal control of pyruvate kinase and that phosphorylation and inactivation of this enzyme results predominantly from the action of the cyclic AMP-dependent protein kinase.