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F A Stephenson

Publications and source records attributed to F A Stephenson.

At least 73 records · Page 4Linked to original sources
PubMed1988

Benzodiazepine binding site heterogeneity in the purified GABAA receptor.

The displacement of [3H]flunitrazepam binding activity by ethyl-beta-carboline-3-carboxylate (beta CCE) was studied in both membrane-bound and purified GABAA receptors from adult bovine cerebral cortex, hippocampus and cerebellum. It was found that the best fit for the displacement of benzodiazepine binding in the cerebellar membranes was a single site with IC50 = 0.55 +/- 0.21 nM, whereas the best fit for cortical and hippocampal membranes was a two-site model with respective values of IC50 = 0.2 +/- 0.09 nM (high affinity), IC50 = 21 +/- 6 nM (low affinity) (cortex) and IC50 = 0.25 +/- 0.05 nM, IC50 = 20 +/- 2 nM (hippocampus). These same properties were retained in the purified GABAA receptor from the three brain regions. Thus, we have demonstrated that binding site heterogeneity as defined by the displacement of beta CCE is preserved in purified GABAA receptors and we suggest that this provides evidence for the existence of GABAA receptor isoforms.

Animals↗
PubMed1988

Phase metastability and supercooled metastable state of diundecanoylphosphatidylethanolamine bilayers.

Aqueous dispersons of L-alpha-phosphatidylethanolamine (PE) with identical saturated acyl chains are known to exhibit gel-state metastability. It is also known that the metastability in PE becomes more pronounced with decreasing acyl chain-length. In an attempt to study the metastable phase behavior of PE, we have synthesized diundecanoylphosphatidylethanolamine (diC11PE) and examined its polymorphic phase behavior. A single endothermic transition at 38 degrees C is detected between 10 and 55 degrees C by DSC for the nonheated sample of diC11PE in excess water. An immediate second heating scan done after cooling slowly of the same sample from the liquid-crystalline state shows a smaller endothermic transition at a lower temperature, 18 degrees C. However, the high-temperature transition at 38 degrees C can be detected, if the sample which has been heated above 38 degrees C is quench cooled from the liquid-crystalline to a temperature between 18 and 38 degrees C. Furthermore, two endothermic transitions at 18 and 38 degrees C and an exothermic transition at 19 degrees C are recorded for diC11PE after quench supercooling of the sample from the liquid-crystalline state to an appropriate temperature below 10 degrees C. The gel-state metastability of diC11PE can be most appropriately explained in terms of changes in interbilayer headgroup-headgroup interactions. It is suggested that the kinetically trapped supercooled metastable state may be a multilamellar structure with melted acyl chains but with strong interbilayer headgroup-headgroup interactions.

Calorimetry↗
PubMed1987

Binary mixtures of asymmetric phosphatidylcholines with one acyl chain twice as long as the other.

High-resolution differential scanning calorimetry and 31P NMR spectroscopy have been used to study aqueous phosphatidylcholine (PC) dispersions prepared from colyophilized mixtures of C(10):C(22)PC/C(22):C(12)PC of various molar ratios. These two lipid species are highly asymmetric but have a common structural feature; namely, one acyl chain in the fully extended conformation is about twice as long as the other. Our experimental results support two conclusions: (1) These two component lipids are miscible in all proportions in both gel and liquid-crystalline states. This type of system behaves as a nearly ideal mixture. Its calorimetric parameters are those expected on the basis of the mole fraction weighted average of the corresponding parameters for the pure components. (2) The component lipids appear to self-assemble, at T less than Tm, into a mixed interdigitated bilayer in excess water. In a mixed interdigitated bilayer, the short acyl chain of one asymmetric phosphatidylcholine on one side of the bilayer leaflet is apposed with the short acyl chain of another lipid molecule on the other side of the bilayer leaflet, while the longer acyl chain from each of the two leaflets crosses the entire hydrocarbon width of the bilayer. The fundamental packing unit, as well as the dynamic unit describing the axial rotator motion about the bilayer normal for this mixed interdigitated bilayer, is thus a dimer, whereas the packing unit assigned for the noninterdigitated bilayer such as C(16):C(16)PC lamellae is a monomer.

Calorimetry, Differential Scanning↗
PubMed1987

The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

The GABAA receptor has been purified to homogeneity from bovine cerebral cortex. Under stringent conditions of isolation, the GABAA receptor was shown to consist only of alpha (Mr 53 000) and beta (Mr 57 000) subunits. A densitometric scan of SDS-PAGE gels under reducing conditions showed that these subunits were present in a 1:1 ratio. A model of the receptor as a heterologous tetramer alpha 2 beta 2 is proposed. Monoclonal antibodies have been raised to the purified bovine GABAA receptor. One of these antibodies, 1A6, was shown to react with both the alpha and beta subunits of the purified receptor. The subunits were still positive in immunoblots following the removal of the carbohydrate moieties of the respective polypeptides by endoglycosidase F treatment. This antibody has been employed to demonstrate antigenic cross-reactivity between the GABAA receptors of three vertebrate species. It is further proposed that there is partial amino acid sequence homology between the alpha and beta polypeptides and hence that they are derived from a single ancestral gene.

Animals↗
PubMed1987

Progress towards the understanding of the GABAA receptor structure.

The GABA receptor of mammalian brain is a ligand-gated channel protein with allosteric binding sites for the benzodiazepines and barbiturate drugs. The receptor is an acidic oligomeric membrane glycoprotein and it has been purified to homogeneity from bovine cerebral cortex, bovine cerebellum and rat cerebral cortex by benzodiazepine affinity chromatography. In each case, extraction and purification with the zwitterionic detergent CHAPS and exogenous phospholipid has demonstrated the coexistence of GABA, benzodiazepine and cage convulsant ligand binding sites on a single protein complex; in addition the allosteric interactions between these sites are preserved in the isolated protein. The receptor has a heterologous structure that is conserved at the subunit level between the aforementioned mammalian species and brain regions. SDS-PAGE has shown that the receptor consists of two subunits, alpha (Mr 53000) and beta (Mr 57000) present in equal stoichiometry. A model consistent with the determination of the molecular weight of the native protein, i.e., Mr 230,000, is that of a tetramer alpha 2 beta 2. [3H]Flunitrazepam and [3H]muscimol have been employed as photoaffinity labels to map the benzodiazepine and GABA binding polypeptides respectively. Polyclonal and monoclonal antibodies have been raised to the native bovine GABAA receptor and these have been employed for the further characterisation of the receptor protein.

Allosteric Site↗
PubMed1986

Separate subunits for agonist and benzodiazepine binding in the gamma-aminobutyric acidA receptor oligomer.

The gamma-aminobutyric acidA (GABAA) agonist muscimol can be photoactivated by 254 nm illumination to affinity label its binding site in the GABAA receptor. We have conducted this reaction on the pure receptor from bovine cerebral cortex in detergent solution, showing that [3H]muscimol can produce then a specific saturable labeling. In the detergent solution, the receptor alone is sensitive to 254 nm irradiation; this reduces the efficiency of incorporation to below that in the membranes, but the competing photoreaction with [3H]muscimol is sufficient and occurs at a representative set of the muscimol-binding sites, such that it can be employed for the photolabeling of those sites. The affinity of [3H]muscimol displayed in this irreversible reaction is indistinguishable from that of its reversible binding. gamma-Aminobutyric acid and bicuculline compete in the photolabeling reaction according to their known affinities at the gamma-aminobutyric acid-binding site. The labeling is shown to occur at the beta-subunit (apparent Mr 57,000) in the pure receptor. The binding sites for gamma-aminobutyric acid agonists, on the beta-subunits, and the benzodiazepine binding sites, on the alpha-subunits, are linked allosterically so that a strongly cooperative hetero-oligomeric structure of this receptor is deduced.

Animals↗
PubMed1986

Polymorphic phase behavior of platelet-activating factor.

Vibrational Raman and 31P NMR spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of 1-0-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine, a chemically synthesized platelet-activating factor. In the temperature range of -7 to 30 degrees C, the C(18)/PAF-H2O system is shown, upon heating, to undergo two thermal phase transitions centered at 9.2 degrees and 18.4 degrees C. The low temperature transition, attributed to the interdigitated lamellar gel (II)----gel (I) phase transition, is characterized by the breakdown of large lamellar organizations into small, but aggregated, bilayer vesicles. The high-temperature transition corresponds to the interdigitated lamellar gel (I)----micellar transition. The molecular ordering and packing structure of C(18)/PAF in the two lamellar phases and phase transition regions are described. It appears that the interdigitated lamellar gel (I) phase is unique for C(18)/PAF dispersions when compared with the behavior of other chemically closely related phospholipids in excess water.

Gels↗
PubMed1986

Antibodies recognising the GABAA/benzodiazepine receptor including its regulatory sites.

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.

Allosteric Regulation↗
PubMed1986

The GABAA receptor and its antibodies.

The GABAA/benzodiazepine receptor has been purified to homogeneity from bovine and rat cerebral cortex. Under optimum conditions, the purified receptor has been shown to possess four distinct drug-binding sites, for GABA, benzodiazepine, barbiturate and Cl- channel gating classes of ligands. The receptor is a multi-subunit membrane glycoprotein with an oligomeric size of 230,000 Da. It contains at least two subunits, alpha and beta, the first of which can be photoaffinity-labelled with the benzodiazepine, flunitrazepam. Polyclonal and monoclonal antibodies have been raised to the native receptor and both have been used in an immunological characterization of the receptor protein in detergent extracts and likewise in purified preparations from bovine cerebral cortex.

Animals↗
PubMed1985

Properties of a specific glycolipid transfer protein from bovine brain.

A transfer protein specific for glycolipids has been isolated from bovine brain. As judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the protein is 68% pure and has a molecular weight of 20 000. Three different assays were employed to study the protein's specificity and glycolipid binding properties. The protein transferred several different neutral glycosphingolipids and ganglioside GM1 equally well, but failed to accelerate phosphatidylcholine or sphingomyelin intervesicular movement. The protein's ability to interact with glycolipids was strongly influenced by the physical properties of the matrix phospholipid in which the glycolipids reside. Both the phase state of the phospholipid matrix and bilayer curvature affected glycolipid intervesicular transfer rates. Protein binding to phospholipid vesicles containing either tritium-labeled or pyrene-labeled glucosylceramide could not be demonstrated by density gradient centrifugation or fluorescence energy transfer measurements, respectively. A specific association of the transfer protein for pyrene-labeled glucosylceramide was found when the fluorescence emission of the pyrene excimer-to-monomer ratio was measured suggesting that a portion of the fluorescent glycolipid was being sequestered from the phospholipid vesicles and was binding to the freely soluble protein.

Animals↗
PubMed1984

Anti-insulin-like (diabetogenic) peptides in pituitary extracts: role of oxytocin.

A peptide has been extracted and characterized from whole bovine pituitaries that has anti-insulin-like activities when assayed in rat adipocytes. This peptide has been purified approximately 100,000-fold, is homogeneous by thin-layer chromatography in three separate solvent systems, and shows a single peak by reverse-phase high-performance liquid chromatography. By these chemical criteria, as well as biological activity criteria (14CO2 production from D-[U-14C]glucose and D-[U-14C]glucose incorporation into glycogen in rat adipocytes], the peptide is indistinguishable from oxytocin. It reacts with anti-oxytocin antibody, and has an amino acid composition indistinguishable from purified oxytocin. The relationship between this material and other previously described anti-insulin or diabetogenic peptides is discussed, but it was not possible to conclude that this peptide, which has been purified to homogeneity and constant specific activity, is related to these previously described factors.

Adipose Tissue↗
PubMed1984

Oxytocin: anti-insulin-like effects in isolated fat cells.

Oxytocin has both insulin-like and insulin antagonistic actions in fat cells in vitro. The anti-insulin-like effects of oxytocin in dispersed rat fat cells have been studied. The magnitude of the anti-insulin-like activity varies with the metabolic pathway of glucose utilization; oxidation [14CO2 production], 32%; glycogen synthesis (D-[U-14C] glucose incorporation into glycogen), 77%. In addition, direct inhibition of the activation of fat cell glycogen synthase has been shown. These inhibitory effects depend upon an intact disulfide ring, since the ability of N-ethylmaleimide-reacted oxytocin to inhibit insulin-stimulated processes was reduced by more than 90% when compared to the intact molecule.

Adipose Tissue↗
PubMed1984

delta 5,7,9(11)-Cholestatrien-3 beta-ol: a fluorescent cholesterol analogue.

Structural analysis, a purification scheme and stability information on a fluorescent cholesterol analogue, which has been used as a probe in several model and biological systems, are presented. The proposed structure for the fluorophore, cholestatrien-3 beta-ol, closely resembles that of cholesterol. However, problems of low yield during synthesis and rapid decomposition have impeded its use. This study concerns the synthesis and purification of cholestatrien-3 beta-ol by reverse phase high performance liquid chromatography (HPLC). Unlike cholestatrien-3 beta-ol recrystallized from solvents, the fluorescent sterol purified by HPLC was stable over several months at -70 degrees C either as a white, crystalline powder or in ethanolic solution. In model membranes the fluorescence of cholestatrien-3 beta-ol was stable to ultraviolet (UV) light. A simple spectroscopic assay for purity is presented. Included are detailed absorbance, fluorescence, mass, 1H-NMR, and 13C-NMR spectral analyses. The data confirm the structure of cholestatrien-3 beta-ol proposed, but not proven, over 50 years ago, delta 5,7,9(11)-cholestatrien-3 beta-ol.

Cholestenes↗
PubMed1984

The purified GABA/benzodiazepine/barbiturate receptor complex: four types of ligand-binding sites, and the interactions between them, are preserved in a single isolated protein complex.

A GABA/benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABAA agonist [3H]muscimol and for the chloride ion channel blocking agent [35S]t-butylbicyclophosphorothionate (TBPS), whereas the binding properties for the benzodiazepine [3H]flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53 000 and Mr 57 000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [3H]flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [3H]flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53 000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48 000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires greater than 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments less than Mr 10 000.

Animals↗