ABSTRACT
Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.
Subject(s)
Cell Membrane/metabolism , Digitonin/pharmacology , Luteal Cells/metabolism , Progesterone/metabolism , Receptors, Progesterone/metabolism , Animals , Cattle , Cholesterol/chemistry , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Digitonin/chemistry , Digitonin/isolation & purification , Female , Hemolysis , Mass Spectrometry , Pregnenolone/chemistry , Rats , Rats, Wistar , TritiumSubject(s)
ATP-Binding Cassette Transporters , Adenosine Triphosphate/metabolism , Glyburide/chemical synthesis , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Animals , Binding Sites , CHO Cells , COS Cells , Chromatography, Affinity/methods , Cricetinae , Digitonin/isolation & purification , Glyburide/analogs & derivatives , Iodine Radioisotopes , Photoaffinity Labels , Potassium Channels/isolation & purification , Receptors, Drug/isolation & purification , Sulfonylurea ReceptorsABSTRACT
Digitonin is widely used for extracting active neurotransmitter receptors from membranes. However, its low critical micellar concentration has made its removal from samples problematic. Here we report that digitonin can be efficiently removed (> 90%) from solution using Extracti-Gel D, a detergent-absorbing matrix. Active kappa 1 opioid receptors solubilized from brain survive Extracti-Gel D chromatography with a recovery of 50-55% and 25% dilution by added volume. The loss of receptor and the dilution, however, are compensated for to a large extent by the disinhibition of binding that results from the removal of digitonin. Extracti-Gel D chromatography had little or no effect on the apparent equilibrium dissociation constant for [3H]U-69,593 binding to the kappa 1 receptor. We conclude that Extracti-Gel D column chromatography is a simple, highly efficient and practical method for markedly reducing the concentration of digitonin in biological samples. Application of the procedure should allow characterization of digitonin-solubilized receptors with minimal complications from bound digitonin and extend the usefulness of digitonin to studies going beyond the initial stages of receptor purification.
Subject(s)
Benzeneacetamides , Digitonin/isolation & purification , Receptors, Opioid, kappa/isolation & purification , Absorption , Analgesics/pharmacokinetics , Animals , Brain Chemistry , Chromatography, Ion Exchange , Guinea Pigs , Kinetics , Membranes/chemistry , Nerve Tissue Proteins/analysis , Pyrrolidines/pharmacokinetics , SolventsABSTRACT
A method for the separation of 3beta-hydroxysterols from other sterols is presented. The method involves precipitating 3beta-hydroxysterols as the digitonides. The digitonide is then decomposed and separated into components using chromatography on highly cross-linked lipophilic polysaccharide gels. The digitonin in the mother liquor is separated from other sterols using the same chromatographic procedure.