Search PubMed⌕ Search

Biomedical subjects

F A Stephenson

Publications and source records attributed to F A Stephenson.

87 records · Page 5Linked to original sources
PubMed1983

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.

Animals↗
PubMed1982

Physicochemical characterization of detergent-solubilized gamma-aminobutyric acid and benzodiazepine receptor proteins from bovine brain.

[3H]Muscimol and [3H]flunitrazepam binding activities have been solubilized from bovine cortex using the ionic detergent sodium deoxycholate. The soluble receptor proteins were shown to bind [3H]muscimol with a dissociation constant, Kd, of 12 nM and a binding capacity (Bmax value) of 1.56 pmol/mg protein; gamma-amino[3H]-butyric acid with a Kd of 50 nM and Bmax of 1.55 pmol/mg protein; and [3H]flunitrazepam with a Kd of 8 nM and a Bmax of 0.8 pmol/mg protein. Gel filtration of the soluble receptor proteins showed that the gamma-amino[3H]butyric acid and [3H]flunitrazepam binding activities comigrated with a Stokes radius of 6.8 nm. The two binding activities were also found to comigrate after sedimentation in a sucrose density gradient. The hydrodynamic properties of the assumed protein-detergent complexes were determined by gel filtration and sedimentation through gradients of sucrose in H2O or 2H2O. Under the conditions employed, the parameters for both the putative gamma-aminobutyric acid and benzodiazepine receptors were: partial specific volume, 0.73 ml g-1; sedimentation coefficient, 12.5 S; molecular weight, 355000; and frictional ratio 1.46. These observations are consistent with the conclusion that the majority of both binding activities solubilized in deoxycholate reside in a single macromolecular complex. However, Triton X-100 selectively solubilized the benzodiazepine binding activity. This suggests that the two binding activities can be at least partially separated.

Animals↗
PubMed1982

Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex.

Barbiturates enhance the binding of [3H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, KD, for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [3H]Flunitrazepam binding activity is stable with storage at 4 degrees C, but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly (t 1/2 = 48 h). [3H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.

Animals↗
PubMed1981

The isolation and characterisation of the nicotinic acetylcholine receptor from human skeletal muscle.

Nicotinic acetylcholine receptor protein has been purified from human skeletal muscle by a procedure involving extraction in non-ionic detergent followed by affinity purification on immobilised alpha-toxin. Purified receptor preparations had specific activities of 0.5-3.5 mumol alpha-bungarotoxin binding sites/g protein and sedimented as a single 125I-alpha-bungarotoxin-binding species in sucrose-density-gradient centrifugation with s20,w = 9.5 S. The purified protein focussed as a single sharp band at pH 5.1 when complexed to 125I-alpha-bungarotoxin. Polyacrylamide gel electrophoresis of the purified receptor under denaturing conditions showed two major protein bands with Mr 42 000 and 66 000 respectively with the occasional appearance of minor components of Mr 56 000 and 85 000. Only the 42 000-Mr band was labelled with the affinity reagent, 4-(N-maleimido)[3H]-benzyltrimethylammonium. The purified receptor bound 125I-alpha-bungarotoxin and d-tubocurarine with Kd values of 0.5 nM and 0.25 microM respectively. It behaved similarly to unpurified detergent-extracted human receptor in the radioimmunoassay for anti-(human acetylcholine receptor) antibodies and when injected into rabbits caused increased levels of the latter antibodies but did not cause experimental autoimmune myasthenia gravis.

Animals↗
PubMed1981

Human urinary insulin-potentiating peptide: immunological evidence for the absence of the human growth hormone amino terminal sequence, Phe-Pro-Thr-Ile-Pro-.

The immunological reactivities of synthetic partial sequences of human growth hormone (hGH) and of an insulin-potentiating peptide isolated from human urine were studied using an antiserum raised against a synthetic hGH analogue [beta-Ala13]hGH(1-15). This antiserum contained antibodies directed mainly against a determinant in the sequence hGH(1-5). It was found that synthetic peptides containing the sequence Phe-Pro-Thr-Ile-Pro- (residues 1-5 of hGH) were immunologically active. Of the biologically active peptides studied, the synthetic peptide hGH(6-13) was virtually inactive and the urinary peptide was completely inert. Absence of immunologically detectable cross-reactivity lends support to the hypothesis that the amino acid sequence of urinary insulin-potentiating peptide (U-AcG) does not contain the hGH amino terminal sequence, Phe-Pro-Thr-Ile-Pro-.

Amino Acid Sequence↗
PubMed1981

Effect of ammonia on glucose oxidation by isolated rat adipocytes.

The effect of ammonium and acetate salts on glucose metabolism in isolated rat adipocytes was studied. Ammonia in low concentration (1-10 mM) was found to inhibit insulin-stimulated CO2 production. Acetate had no effect on this system except at high concentrations (100 mM), at which it stimulated CO2 production. Ammonium and acetate salts had an anti-lipolytic effect at high concentration (100 mM) but no effect on lipolysis at lower concentrations. The observation that trace amounts of salts can affect a biological assay system suggests that other biological assay system may also be altered by contaminating buffer salts from protein isolation steps.

Adipose Tissue↗
PubMed1981

An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis.

A reproducible radioimmunoassay procedure for the determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is described and examined in detail. The assay combines features of a number of methods previously outlined and allows repeat determinations of antibody titre in a given myasthenic serum sample with coefficient of variation 6%. The mean +/- standard deviation for normal human serum anti-acetylcholine receptor antibodies was found by this procedure to be 0.024 +/- 0.033 nmol/l alpha-bungarotoxin binding sites whereas the range for myasthenic patients was 0-139.14 nmol/l with a mean value of 7.55 nmol/l alpha-bungarotoxin binding sites.

Acetylcholine↗
PubMedDate not supplied

Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family.

Amino-acid sequences derived from complementary DNAs encoding the alpha- and beta-subunits of the GABA/benzodiazepine receptor from bovine brain show homology with other ligand-gated receptor subunits, suggesting that there is a super-family of ion-channel-containing receptors. Co-expression of the in vitro-generated alpha-subunit and beta-subunit RNAs in Xenopus oocytes produces a functional receptor and ion channel with the pharmacological properties characteristic of the GABAA receptor.

Animals↗
PubMedDate not supplied

An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor.

The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied. Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-D-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM). TM treatment resulted in a decrease of the NR1 subunit with M(r) 117 000 with a concomitant increase in a M(r) 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [3H]MK801 binding to the expressed receptor which was a result of an approximate four-fold reduction in the Dissociation Constant (KD) but with no change in the number of binding sites (Bmax). NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples. The removal of TM from the cell culture media resulted in a return to the control KD value for [3H]MK801 binding and partial reglycosylation of newly synthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor.

Animals↗