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Molecular Membrane Biology 2005The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the...
The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT(1A) receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT(1A) receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT(1A) receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT(1A) receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT(1A) receptor function.
Topics: Animals; Cattle; Cholesterol; Digitonin; Hippocampus; Ligands; Membranes; Protein Binding; Receptor, Serotonin, 5-HT1A
PubMed: 16096266
DOI: 10.1080/09687860500093453 -
Vision Research Feb 1977
Topics: Digitalis Glycosides; Digitonin; Humans; Methods; Retinal Pigments; Solutions
PubMed: 867852
DOI: 10.1016/0042-6989(77)90095-5 -
The Biochemical Journal May 1985Glucose-induced insulin secretion is thought to be mediated by submicromolar increases in intracellular Ca2+, although the intracellular processes are not well...
Glucose-induced insulin secretion is thought to be mediated by submicromolar increases in intracellular Ca2+, although the intracellular processes are not well understood. We have used the previously characterized digitonin-permeabilized insulin-secreting pancreatic islet model to study the role of myo-inositol 1,4,5-trisphosphate (IP3), a putative second messenger for mobilization of intracellular Ca2+. Ca2+ efflux from the endoplasmic reticulum was studied with or without vanadate present to inhibit Ca2+ reuptake. IP3 (10 microM), at a free Ca2+ level of 0.06 microM, increased Ca2+ release by 30% and, when vanadate was present, by 50%. Maximal and half-maximal Ca2+ release was observed at 10 microM- and 2.5 microM-IP3, respectively. IP3 provoked a rapid release that was followed by slow reuptake. Reuptake was diminished in the presence of vanadate. Inositol 1,4-bisphosphate, inositol 1-phosphate and other phosphoinositide metabolites did not have any significant effect. Because increases in Ca2+ levels in the submicromolar range have been previously shown to induce insulin release in digitonin-permeabilized islets, our results are consistent with the concept of IP3 serving as a second messenger for insulin secretion.
Topics: Adenosine Triphosphate; Animals; Calcium; Cell Membrane Permeability; Digitonin; Dose-Response Relationship, Drug; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Islets of Langerhans; Male; Models, Biological; Rats; Rats, Inbred Strains; Sugar Phosphates
PubMed: 3890834
DOI: 10.1042/bj2270965 -
Methods in Enzymology 1988
Topics: Adipose Tissue; Animals; Cell Membrane Permeability; Cyclic AMP; Digitonin; Kinetics; Lipolysis; Rats
PubMed: 2842586
DOI: 10.1016/0076-6879(88)59020-1 -
Biochemical Pharmacology Aug 1983The effect of the calmodulin antagonist trifluoperazine on fluid phase, adsorptive and receptor-mediated pinocytosis in cultured human fibroblasts and mouse peritoneal...
The effect of the calmodulin antagonist trifluoperazine on fluid phase, adsorptive and receptor-mediated pinocytosis in cultured human fibroblasts and mouse peritoneal macrophages was studied. Uptake in each case was reversibly inhibited by about 20% without toxic effects. Low concentrations of the detergent digitonin had a similar effect. Thus the three uptake mechanisms have common features, and we suggest that in both fibroblasts and macrophages they may reflect uptake within vesicles participating in receptor-mediated endocytosis. These effects of trifluoperazine may operate directly on membranes without the participation of calmodulin.
Topics: Animals; Calmodulin; Digitonin; Fibroblasts; Humans; Macrophages; Mice; Pinocytosis; Trifluoperazine
PubMed: 6615543
DOI: 10.1016/0006-2952(83)90685-8 -
Photochemistry and Photobiology 2005Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments...
Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20 degrees C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.
Topics: Animals; Cattle; Digitonin; Kinetics; Rhodopsin; Rod Cell Outer Segment; Spectrophotometry
PubMed: 15819603
DOI: 10.1562/2005-02-10-RA-437 -
Journal of Neurochemistry Nov 1992The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding...
The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.
Topics: Animals; Brain Chemistry; Cattle; Digitonin; GTP-Binding Proteins; Molecular Weight; Receptors, Histamine; Receptors, Histamine H3; Solubility
PubMed: 1328529
DOI: 10.1111/j.1471-4159.1992.tb10996.x -
Molecular and Biochemical Parasitology Aug 1993Mitochondrial ATPase of Leishmania donovani was characterized using digitonin-permeabilized promastigotes and the results were compared with those from isolated...
Mitochondrial ATPase of Leishmania donovani was characterized using digitonin-permeabilized promastigotes and the results were compared with those from isolated mitochondria. Maximum mitochondrial ATPase activity was obtained in promastigotes permeabilized with digitonin at a final concentration of 20 microM and the specific activity of the enzyme was 46% and 57% higher than that of homogenized and sonicated promastigotes, respectively. At concentrations above 20 microM digitonin inhibited ATPase activity and the degree of inhibition increased with increasing concentrations of the detergent. The ATPase activity of promastigotes remained DCCD-sensitive when permeabilized with digitonin at concentrations up to 120 microM but the enzyme became increasingly resistant to this inhibitor as digitonin concentrations were increased to 140 microM and more, indicating the loss of functional activity of the enzyme. The pH and temperature optima for mitochondrial ATPase were determined to be 7.5 and 30 degrees C, respectively. Mg2+ ions were essential for ATPase activity but free Mg2+ ions were found to be inhibitory. A Mg2+/ATP ratio of 1:3 supported the optimum ATPase activity. Sulfite and hexanol activated the enzyme but failed to prevent the inhibition by free Mg2+ ions. The results indicate that digitonin-permeabilized promastigotes provide an ideal system for studying the mitochondrial ATPase of L. donovani.
Topics: Adenosine Triphosphatases; Alcohols; Animals; Cell Membrane Permeability; Digitonin; Hydrogen-Ion Concentration; Ions; Leishmania donovani; Magnesium; Mitochondria; Oligomycins
PubMed: 8232420
DOI: 10.1016/0166-6851(93)90140-s -
Journal of Neuroscience Methods Jul 1992A simple and highly sensitive assay for measuring total digitonin in biological samples is described. The assay is based on the ability of digitonin to hemolyze red...
A simple and highly sensitive assay for measuring total digitonin in biological samples is described. The assay is based on the ability of digitonin to hemolyze red blood cells. The precision and reproducibility of the assay was excellent with intra- and inter-assay variabilities of less than 1% and 6%, respectively. The assay was used to evaluate several potential methods for removing digitonin from biological samples (digitonin extracts from guinea pig brain membranes). Dialysis and G-25 Sephadex chromatography were ineffective. However, protein and digitonin can be effectively separated by ammonium sulfate precipitation followed by dialysis. The kappa 1 opioid receptor survived these procedures with no change in affinity for [3H]U-69,593. In conclusion, the hemolytic assay for digitonin appears to provide a practical means for determining detergent concentrations during receptor purification and characterization and for evaluating potential methods for detergent removal. Although an in depth analysis of the assay was carried out only for digitonin, CHAPS and deoxycholate also caused 50% hemolysis at concentrations well below those commonly used for receptor solubilization and, therefore, the general assay procedures might have applicability for measurement of these and perhaps other detergents used in receptor solubilization as well.
Topics: Animals; Brain Chemistry; Chromatography, Ion Exchange; Dialysis; Digitonin; Guinea Pigs; Hemolysis; In Vitro Techniques; Membranes; Rats; Receptors, Cell Surface; Solubility
PubMed: 1328773
DOI: 10.1016/0165-0270(92)90024-8 -
The Journal of General Virology Nov 2007Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membrane in vivo, nucleocapsids are believed to...
Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membrane in vivo, nucleocapsids are believed to gain entry into the cytosol via a membrane fusion event involving the inner membrane and the plasma membrane of susceptible host cells; accordingly, experimentally induced damage to the outer membrane might be expected to increase infectivity. Here, in an attempt to develop an in vitro model system for studying ichnovirus infection, we show that digitonin-induced disruption of the virion outer membrane not only increases infectivity, but also uncovers an activity not previously associated with any polydnavirus: fusion from without.
Topics: Animals; Cell Line; Digitonin; Polydnaviridae; Spodoptera; Virion; Virus Internalization; Viruses
PubMed: 17947519
DOI: 10.1099/vir.0.83118-0